IL296312A

IL296312A - Methods, systems and kits for in-pen assays

Info

Publication number: IL296312A
Application number: IL296312A
Authority: IL
Original assignee: Berkeley Lights Inc
Priority date: 2020-03-09
Filing date: 2021-03-08
Publication date: 2022-11-01
Also published as: WO2021183458A3; TW202201008A; JP2023517583A; AU2021235901A1; WO2021183458A2; EP4118438A4; KR20220166788A; EP4118438A2; US20230251268A1; CN115516318A; CA3170874A1

Description

METHODS, SYSTEMS AND KITS FOR IN-PEN ASSAYS BACKGROUNDThe embodiments disclosed herein are generally directed towards, systems, apparatuses and methods for optically measuring a quantity or quality parameter of a micro-object confined within a defined area. More specifically, there is a need for imaging systems and methods that can accurately determine the quantity of an analyte produced by a micro-object confined in a chamber (e.g., sequestration chamber) within a microfluidic device. SUMMARY In one aspect ,a system is provided for determining a quantity of analyte produced by a biological micro-object. The system can comprise an image acquisition unit. The image acquisition unit can comprise a microfluidic device holder capable of securing a microfluidic device, wherein the microfluidic device includes a flow region and a plurality of chambers that are fluidically connected to the flow region. Each of the plurality of chambers can hold one or more biological micro-objects. The image acquisition unit can further comprise an imaging element configured to capture one or more assay images of the plurality of chambers and the flow region of the microfluidic device. The system can further comprise an image processing unit communicatively connected to the image acquisition unit. The image processing unit can comprise an area of interest determination engine configured to receive each captured assay image and define an area of interest for each chamber depicted in the assay image. The area of interest can include an image area corresponding to an area within the chamber that is most sensitive for measuring analyte concentration fluctuations, is least sensitive to the position of biological micro-objects in the chamber when analyte fluctuations are measured, and/or extends along an axis of diffusion between the chamber and the flow region. The image processing unit can further comprise a scoring engine configured to analyze at least a portion of the image area within the area of interest of each chamber, to determine scores that are indicative of the quantity of analyte in each chamber. In another aspect, a method is provided for determining a quantity of analyte produced by a biological micro-object. The method can comprise the step of receiving imaging data of a microfluidic device that includes a flow region and a plurality of chambers that are fluidically connected to the flow region. The imaging data can include an analyte assay image and one or both of a background noise image and a signal reference image. The method can further comprise defining an area of interest for each chamber. The area of interest can include an image area within the chamber that is most sensitive for measuring analyte concentration fluctuations, is least sensitive to the position of biological micro-objects in the chamber when analyte fluctuations are measured, and/or extends along an axis of diffusion between the chamber and the flow region. The method can even further comprise determining scores (e.g., scores that provide a relative ranking of chambers across the chip), where the score is indicative of the quantity of analyte in each chamber by analyzing at least a portion of the image area of the area of interest for each chamber. In another aspect, a non-transitory computer-readable medium is provided in which a program is stored for causing a computer to perform an image processing method for determining a quantity of analyte produced by a biological micro-object. The method can comprise receiving imaging data of a microfluidic device that includes a flow region and a plurality of chambers that are fluidically connected to the flow region. The imaging data can include an analyte assay image and one or both of a background noise image and a signal reference image. The method can further comprise defining an area of interest for each chamber. The area of interest can include an image area within the chamber that is most sensitive for measuring analyte concentration fluctuations, is least sensitive to the position of biological micro-objects in the chamber when analyte fluctuations are measured, and/or extends along an axis of diffusion between the chamber and the flow region. The method can even further comprise determining scores that are indicative of the quantity of analyte in each chamber by analyzing at least a portion of the image area of the area of interest for each chamber. In another aspect, a method of assessing a level of secretion of an analyte by a biological micro-object, or a population of biological micro-objects generated therefrom is provided, the method comprising introducing the biological micro-object into a chamber of a microfluidic device, wherein the microfluidic device includes an enclosure having a flow region, where the chamber is fluidically connected to the flow region, and wherein chamber contains a first fluidic medium; allowing the biological micro-object, or the population of biological micro-objects generated therefrom, to secrete the analyte into the first fluidic medium within the chamber; introducing a second fluidic medium into the flow region, wherein the second fluidic medium contains a plurality of soluble reporter molecules, and where each soluble reporter molecule includes a binding component configured to bind the secreted analyte and a detectable label; allowing a portion of the plurality of soluble reporter molecules to diffuse into the chamber and bind to the analyte secreted therein, thereby producing a plurality of soluble reporter molecule: secreted analyte (RMSA) complexes; and detecting soluble reporter molecules and/or soluble RMSA complexes located within an area of interest within the microfluidic device, wherein the area of interest includes at least a portion of the chamber. In another aspect, a method of clonal line development is provided, the method including: introducing an individual biological micro-object into each of a plurality of chambers of a microfluidic device, where the microfluidic device further includes an enclosure having a flow region, and where each of the chambers of the plurality is fluidically connected to the flow region and contains a first fluidic medium; allowing each biological micro-object, or a clonal population of biological micro-objects generated therefrom, to secrete an analyte into the first fluidic medium contained in the corresponding chamber; introducing a second fluidic medium into the flow region, where the second fluidic medium includes a plurality of soluble reporter molecules, where each soluble reporter molecule includes a binding component configured to bind the secreted analyte and a detectable label; allowing a portion of the plurality of soluble reporter molecules to diffuse into each chamber of the plurality and bind to at least a portion of the analyte secreted therein, thereby producing a plurality of soluble reporter molecule:secreted analyte (RMSA) complexes in each of the plurality of chambers; detecting, for each chamber of the plurality, an intensity of a signal emanating from a corresponding area of interest, where the area of interest includes at least a portion of the corresponding chamber, and where at least a portion of the signal emanating from the area of interest emanates from the detectable label of soluble reporter molecules and/or soluble RMSA complexes located within the area of interest; determining, for each chamber of the plurality, a score based upon the detected signal intensity emanating from the corresponding area of interest; selecting a set of chambers from the plurality of chambers, where each chamber of the set has a score indicating that the biological micro-object, or clonal population, contained therein is a top analyte producer; and exporting from the microfluidic device one or more biological micro-objects contained within each chamber of the set of selected chambers. In certain embodiments, the method can further comprise expanding the exported one or more biological micro-objects from each chamber of the set of selected chambers to form clonal populations in corresponding reaction vessels; and determining a level of analyte secreted in each corresponding reaction vessel, thereby determining a level of secretion for each clonal population. In yet another aspect, a kit for evaluation of secretion levels of analyte of a biological micro- object or a population of biological micro-objects generated therefrom is provided, including: a microfluidic device including an enclosure having a flow region and a chamber, where the chamber is fluidically connected to the flow region, and where the flow region and the chamber are configured to contain a fluidic medium; and a soluble reporter molecule comprising a detectable label and a binding component configured to bind the analyte. In certain embodiments, the soluble reporter molecule is provided in solution. In other embodiments, the soluble reporter molecule is provided in a dry (e.g., lyophilized) form. In various embodiments, a method is providing for assessing a level of secretion of an analyte by a biological micro-object, or a population of biological micro-objects generated therefrom, the method comprising: introducing the biological micro-object into a chamber of a microfluidic device, wherein the microfluidic device comprises an enclosure having a flow region, wherein the chamber is fluidically connected to the flow region, and wherein the chamber contains a first fluidic medium; allowing the biological micro-object, or the population of biological micro-objects generated therefrom, to secrete an analyte into the first fluidic medium within the chamber; introducing a second fluidic medium into the flow region, wherein the second fluidic medium comprises a plurality of soluble reporter molecules, and wherein each soluble reporter molecule comprises a binding component configured to bind the secreted analyte and a detectable label; allowing a portion of the plurality of soluble reporter molecules to diffuse into the chamber and bind to the analyte secreted therein, thereby producing a plurality of soluble reporter molecule: secreted analyte (RMSA) complexes; introducing a third fluidic medium into the flow region, wherein the third fluidic medium does not comprise any of the soluble reporter molecules; allowing at least a portion of unbound reporter molecules to diffuse out of the chamber; and detecting soluble reporter molecules and/or soluble RMSA complexes located within an area of interest within the microfluidic device, wherein the area of interest is within an unswept region of the chamber. In various embodiments, a method is provided of assessing a level of secretion of an analyte by a biological micro-object, or a population of biological micro-objects generated therefrom, the method comprising: introducing a first fluidic medium into the flow region, wherein the first fluidic medium comprises a plurality of soluble reporter molecules, and wherein each soluble reporter molecule comprises a binding component configured to bind the secreted analyte and a detectable label; measuring the intensity of the detectable signal within an area of interest; introducing a second fluidic medium into the flow region, wherein the second fluidic medium does not comprise any of the soluble reporter molecules; introducing the biological micro-object into a chamber of a microfluidic device, wherein the microfluidic device comprises an enclosure having a flow region, wherein the chamber is fluidically connected to the flow region; allowing the biological micro-object, or the population of biological micro-objects generated therefrom, to secrete an analyte into the first fluidic medium within the chamber; introducing a third fluidic medium into the flow region, wherein the second fluidic medium comprises a plurality of the soluble reporter molecules, and wherein each soluble reporter molecule comprises a binding component configured to bind the secreted analyte and a detectable label; allowing a portion of the plurality of soluble reporter molecules to diffuse into the chamber and bind to the analyte secreted therein, thereby producing a plurality of soluble reporter molecule: secreted analyte (RMSA) complexes; and detecting soluble reporter molecules and/or soluble RMSA complexes located within an area of interest within the microfluidic device.

In various embodiments, a method is provided, wherein the area of interest comprises at least a portion of the chamber aligned along an axis of diffusion from within the chamber out into the flow region. In various embodiments, a method is provided wherein the area of interest is within an unswept region of the chamber. In various embodiments, a method is provided further comprising introducing a third fluidic medium into the flow region, wherein the third fluidic medium does not comprise any of the soluble reporter molecules; allowing at least a portion of unbound soluble reporter molecules to diffuse out of the chamber; and detecting soluble reporter molecules and/or soluble RMSA complexes located within the area of interest within the microfluidic device. In various embodiments, a method is provided wherein detecting the soluble reporter molecules located within the area of interest occurs at a time selected such that an amount of unbound soluble reporter molecules that have diffused out of the chamber is at least 2X greater than an amount of soluble RMSA complexes that have diffused out of the chamber. In various embodiments, a method is provided wherein the method further comprises quantifying the level of secretion in an analyte. In various embodiments, a method is provided wherein the secreted analyte has a molecular weight at least twice as great as a molecular weight of the soluble reporter molecules. In various embodiments, a method is provided wherein the secreted analyte has a molecular weight at least four times greater than a molecular weight of the soluble reporter molecules. In various embodiments, a method is provided wherein the secreted analyte has a molecular weight at least ten times greater than a molecular weight of the soluble reporter molecules. In various embodiments, the analyte secreted by the biological micro-object comprises a protein, a saccharide, a nucleic acid, an organic molecule other than a protein, saccharide, or nucleic acid, a vesicle, or a virus. In various embodiments, the analyte secreted by the biological micro-object is an antibody or, optionally, a glycosylated antibody. In various embodiments, the biological micro-object is a biological cell. In various embodiments, the binding component of the soluble reporter molecule comprises at least one amino acid and/or at least one nucleic acid. In various embodiments, the binding component of the soluble reporter molecule comprises a peptide or protein. In various embodiments, the binding component of the soluble reporter molecule comprises a peptide having the sequence of any one of SEQ ID NOs: 1 to 10.

In various embodiments, the binding component of the soluble reporter molecule comprises protein A, protein G, or an IgG-binding fragment of protein A or protein G. In various embodiments the binding component of the soluble reporter molecule comprises an aptamer. In various embodiments, the detectable label comprises a visible, luminescent, phosphorescent or fluorescent label. In various embodiments the detectable label of the soluble reporter molecules is a fluorescent label, and wherein said detecting the soluble reporter molecules and/or soluble RMSA compleses comprises detecting fluorescence emission from the fluorescent label of the soluble reporter molecules and/or soluble RMSA complexes within the area of interest. In various embodiments, a method is provided wherein said detecting fluorescence emission is performed after a second period of time; said detecting fluorescence emission is performed two or more times during a second period of time; or said detecting fluorescence emission is performed substantially continuously during a second period of time. In various embodiments, a method is provided further comprising exposing, for a third period of time, a portion of the microfluidic device comprising the chamber to electromagnetic radiation comprising a wavelength which is capable of exciting the fluorescent label of the soluble reporter molecules. In various embodiments, a method is provided further comprising exposing, for a fourth period of time, a portion of the microfluidic device comprising the chamber but not the flow region, wherein the fourth period of time is sufficient to photobleach the fluorescent label of any soluble reporter molecules and/or soluble RMSA complexes present in the chamber. In various embodiments, said exposing for a fourth period of time is performed before said detecting fluorescence emission within the area of interest. In various embodiments said detecting fluorescence emission within the area of interest occurs about 5 seconds to about 20 seconds after said exposing for a fourth period of time (e.g., after reporter molecules comprising non-photobleached fluorescent label have diffused into said chamber). In various embodiments, the method further comprises perfusing the flow region with a culturing medium, wherein the perfusing occurs after introducing the biological micro-object into the chamber and before introducing the second fluidic medium into the flow region. In various embodiments, the culturing medium comprises one or more of a soluble feeder cell component, a defined dissolved oxygen component, defined pH component, an exhausted growth medium component, and/or a soluble stimulatory component.

In various embodiments, introducing the second fluidic medium into the flow region comprises flowing the second fluidic medium through the flow region for a first period of time. In various embodiments the first period of time is about 30 to about 60 minutes. In various embodiments, detecting soluble reporter molecules and/or soluble RMSA complexes located within the area of interest comprises measuring an intensity of a detectable signal coming from the area of interest, wherein at least some of the detectable signal emanates from the detectable label of soluble reporter molecules and/or soluble RMSA complexes located within the area of interest. In various embodiments, detecting the soluble reporter molecules located within the area of interest further comprises determining a background-subtracted signal intensity by subtracting an intensity of a background signal from the measured intensity of the detectable signal. In various embodiments, the measured intensity of the detectable signal or the background-subtracted signal intensity is normalized for a number of cells observed within the chamber. In various embodiments, the method further comprises providing a secretion score for the chamber. In various embodiments, the secretion score is determined according to the method of any one of embodiments 12 to 25 (i.e., from the Recitation of Selected Embodiments). In various embodiments, the microfluidic device comprises a plurality of chambers, wherein a biological micro-object is introduced into at least two chambers of the plurality, and wherein the remainder of the method is carried out with respect to each of the at least two chambers. In various embodiments, the method further comprises comparing a level for chambers of the at least two chambers of the plurality. In various embodiments, the method further comprises comparing secretion scores of more than one chamber of the plurality of chambers. In various embodiments, the method further comprises selecting one or more of the at least two chambers; and exporting one or more biological micro-objects from each of the selected chambers. In various embodiments, the one or more biological micro-objects from each of the selected chambers are further exported out of the microfluidic device. In various embodiments, the selected chambers are exported individually. In various embodiments, the area of interest comprises an image area corresponding to an area within the chamber that is most sensitive for measuring analyte concentration fluctuations, and/or is least sensitive to the position of biological micro-objects in the chamber when analyte fluctuations are measured.

In various embodiments the area of interest comprises an image area corresponding to an area within the chamber that is most sensitive for measuring. In various embodiments the area of interest consists essentially of the image area. In various embodiments the method is automated. In various embodiments, a method is provided for clonal cell line development, the method comprising the performing any of the methods described above, selecting a set of chambers from the plurality of chambers, wherein each chamber of the set has a score indicating that the biological micro-object, or clonal population, contained therein is a top analyte producer; exporting from the microfluidic device one or more biological micro-objects contained within each chamber of the set of selected chambers; expanding the exported one or more biological micro-objects from each chamber of the set of selected chambers in corresponding reaction vessels; and determining a level of analyte secreted in each corresponding reaction vessel, thereby determining a level of secretion for each biological micro-object or clonal population. In various embodiments the area of interest comprises an image area corresponding to an area within the chamber that is most sensitive for measuring analyte concentration fluctuations, is least sensitive to the position of biological micro-objects in the chamber when analyte fluctuations are measured, and/or extends along an axis of diffusion between the chamber and the flow region. In various embodiments the method is automated. In various embodiments, a non-transitory computer-readable medium is provided in which a program is stored for causing a computer to direct a system to perform a method for determining a quantity of analyte produced by a biological micro-object, wherein the method is any one of the methods described above. In various embodiments, a non-transitory computer readable medium is provided in which a program is stored for causing a computer to direct a system to perform at least part of a method for clonal line development, wherein the method is the method of any one the claims above, and wherein the system performs at least the steps up until and including exporting from the microfluidic device the one or more biological micro-objects contained within each chamber of the set of selected chambers. In accordance with various embodiments, a method is provided of assessing a relative level of secretion of an analyte across a plurality of biological micro-objects, or across populations of biological micro-objects generated therefrom, the method comprising: introducing a plurality of biological micro-objects into a plurality of chambers of a microfluidic device, wherein the microfluidic device comprises an enclosure having a flow region, wherein each chamber of the plurality of chambers is fluidically connected to the flow region, and wherein the plurality of chambers contains a first fluidic medium; allowing the plurality of biological micro-objects, or the populations of biological micro-objects generated therefrom, to secrete a soluble analyte into the first fluidic medium within the plurality of chambers; introducing a second fluidic medium into the flow region, wherein the second fluidic medium comprises a plurality of soluble reporter molecules, and wherein each reporter molecule comprises: a binding component configured to bind the secreted analyte; and a detectable label; allowing a portion of the plurality of soluble reporter molecules to diffuse into the plurality of chambers and bind to the soluble analyte secreted therein, thereby producing a plurality of soluble reporter molecule:secreted analyte (RMSA) complexes; and detecting the soluble reporter molecules and/or soluble RMSA complexes located within an area of interest within the microfluidic device. In various embodiments, a method is provided, wherein each chamber of the plurality of chambers comprises an isolation region isolated from secondary flow of the second fluidic medium in the flow region. In various embodiments, a method is provided, wherein each chamber of the plurality of chambers comprises a connection region isolated from direct flow of the second fluidic medium in the flow region. In various embodiments, a method is provided, wherein an individual biological micro-object of the plurality of biological micro-objects is disposed into an individual chamber of the plurality of chambers. In various embodiments, a method is provided, wherein the concentration of soluble reporter molecules is from about 1-10 times a K D of the soluble reporter molecules for the analyte. In various embodiments, a method is provided, wherein the second fluidic medium in the flow region is refreshed at a refresh rate from about 0.1-10 seconds, and the refresh rate is a duration of time to replace an entire volume of fluid in the flow region. In various embodiments, a method is provided, wherein allowing the portion of the plurality of reporter molecules to diffuse into the plurality of chambers comprises allowing the plurality of reporter molecules to attain an equilibrium state across the flow region and the plurality of chambers. In various embodiments, a method is provided, wherein the equilibrium state is attained within 3 hours from the introduction of the second fluidic medium. In various embodiments, a method is provided, further comprising: obtaining a level of secretion or secretion score for each of the plurality of chambers; and ranking the plurality of chambers based on the obtained secretion scores. In various embodiments, a method is provided, wherein the plurality of reporter molecules comprises at least two different reporter molecules, each having a different label.

In various embodiments, a method is provided, wherein each chamber comprises a single opening to the flow region. In various embodiments, a method is provided, wherein the flow region comprises a microfluidic channel and each chamber comprises an isolation region and a connection region fluidically connecting the isolation region to the microfluidic channel, wherein the connection region has a proximal opening to the microfluidic channel and a distal opening to the isolation region. In various embodiments, a method is provided, wherein the area of interest is at least partially within an isolation region of the chamber. In various embodiments, a method is provided, wherein the area of interest is within a hook region of the chamber. In various embodiments, a method is provided, wherein the area of interest is proximal to a region in which a biological micro-object of the plurality of biological micro-objects is located. In various embodiments, a method is provided, wherein the area of interest is along an axis of diffusion between the plurality of chambers and the flow region (e.g., along an axis of diffusion between the isolation region of the plurality of chambers and the microfluidic channel, such as along an axis of diffusion defined by the connection region). In various embodiments, a method is provided, wherein the area of interest is not along an axis of diffusion between the plurality of chambers and the flow region (e.g., not along an axis of diffusion between the isolation region of the plurality of chambers and the microfluidic channel, or not along an axis of diffusion defined by the connection region). In various embodiments, a method is provided, wherein the area of interest comprises an image area corresponding to an area within a chamber of the plurality of chambers that is most sensitive for measuring analyte concentration fluctuations, and/or least sensitive to the position of biological micro-objects in the chamber when analyte fluctuations are measured. In various embodiments, a method is provided, wherein detecting reporter molecules located within the area of interest within the microfluidic device is performed after the diffusion of reporter molecules into the plurality of chambers and the binding of reporter molecules to the analyte secreted therein is at or near steady state conditions. In various embodiments, a method is provided, further comprising: introducing a third fluidic medium into the flow region, wherein the third fluidic medium does not comprise reporter molecules; and allowing at least a portion of unbound soluble reporter molecules to diffuse out of the plurality of chambers. In various embodiments, a method is provided, wherein the plurality of soluble reporter molecules from the second fluidic medium being a first plurality of soluble reporter molecules, and prior to introducing the plurality of biological micro-objects into the plurality of chambers of the microfluidic device, the method further comprising: introducing a fourth fluidic medium into the flow region, wherein the fourth fluidic medium comprises a second plurality of soluble reporter molecules; acquiring an image of the microfluidic device; and introducing a fifth fluidic medium into the flow region, wherein the fifth fluidic medium does not comprise soluble reporter molecules. In various embodiments, a method is provided, wherein the image being a first image acquired at t = 0, the detecting of the soluble reporter molecules comprises acquiring a second image at t = 1, wherein at t = 1 the diffusion of unbound soluble reporter molecules between the flow region and the plurality of chambers has approached a steady state. In various embodiments, a method is provided, wherein the area of interest comprises at least a portion of a connection region of each chamber of the plurality of chambers, the method further comprising: subtracting at least the first image from the second image to produce a corrected image; and analyzing the corrected image to determine a relative amount of soluble RMSA complexes in the area of interest comprising at least a portion of each connection region for at least a portion of the plurality of chambers. In various embodiments, a method is provided, wherein the relative amount of soluble RMSA complexes in the area of interest is used to generate a linear approximation of changes in the amount of soluble RMSA complexes, and a slope of the linear approximation is used to compare production of secreted analyte between chambers. In various embodiments, a method is provided, further comprising: acquiring a background image prior to introducing the fourth fluidic medium; and obtaining the corrected image by subtracting at least the first image and the background image from the second image. In various embodiments, a method is provided, wherein the area of interest is a hook region of each chamber of the plurality of chambers, the method further comprising: subtracting at least the first image from the second image to produce a corrected image; and analyzing the corrected image to quantify a relative amount of soluble RMSA complexes in the hook region for at least a portion of the plurality of chambers, wherein the relative amount of soluble RMSA complexes in the hook region is used to compare production of soluble secreted analyte across chambers. In various embodiments, a method is provided, further comprising: acquiring a background image prior to introducing the fourth fluidic medium; and obtaining the corrected image by subtracting at least the first image and the background image from the second image. In various embodiments, a method is provided, wherein the first plurality and the second plurality of soluble reporter molecules comprises a same label.

In various embodiments, a method is provided, wherein the first plurality and the second plurality of soluble reporter molecules comprises two different reporter molecules each having a different label. In various embodiments, a method is provided, wherein the image being a first image acquired at t = 0, the method further comprising: acquiring a second image of the microfluidic device, before introducing the third fluidic medium into the flow region and after allowing the portion of the plurality of soluble reporter molecules to diffuse into the plurality of chambers and bind to the soluble analyte secreted therein. In various embodiments, a method is provided, wherein the detecting of the reporter molecules comprises acquiring a third image of the microfluidic device. In various embodiments, a method is provided, wherein the area of interest comprises at least a portion of a connection region of each chamber of the plurality of chambers, the method further comprising: subtracting at least the first image from the third image to produce a corrected image; and analyzing the corrected image to quantify an amount of soluble RMSA complexes in each of a plurality of sub-regions of each connection region for at least a portion of the plurality of chambers, wherein the amount of soluble RMSA complexes in a portion of the plurality of sub-regions are used to generate a linear approximation, and a slope of the linear approximation is used to compare production of secreted analyte between chambers. In various embodiments, a method is provided, further comprising: acquiring a background image prior to introducing the fourth fluidic medium; and obtaining the corrected image by subtracting at least the first image and the background image from the third image. In various embodiments, a method is provided, further comprising: acquiring a background image prior to introducing the fourth fluidic medium; and obtaining the corrected image by subtracting at least the first image, the second image, and the background image from the third image. In various embodiments, a method is provided, wherein the area of interest is a hook region of each chamber of the plurality of chambers, the method further comprising: subtracting at least the first image from the third image to produce a corrected image; and analyzing the corrected image to quantify an amount of RMSA complexes in the hook region for at least a portion of the plurality of chambers, wherein the amount of RMSA complexes in the hook region is used to compare production of secreted analyte between chambers. In various embodiments, a method is provided, further comprising: acquiring a background image prior to introducing the first fluidic medium; and obtaining the corrected image by subtracting at least the first image and the background image from the third image.

In various embodiments, a method is provided, further comprising: acquiring a background image prior to introducing the first fluidic medium; and obtaining the corrected image by subtracting at least the first image, the second image, and the background image from the third image. In various embodiments, a method is provided, wherein the second image is used to determine whether a diffusion of unbound reporter molecules between the flow region and the plurality of chambers has reached a steady state. In various embodiments, a method is provided, wherein the secreted analyte has a molecular weight at least twice as great as a molecular weight of the soluble reporter molecules. In various embodiments, a method is provided, wherein the secreted analyte has a molecular weight at least four times greater than a molecular weight of the soluble reporter molecules. In various embodiments, a method is provided, wherein the secreted analyte has a molecular weight at least ten times greater than a molecular weight of the soluble reporter molecule. In various embodiments, a method is provided, wherein the analyte secreted by the biological micro-objects comprises a protein, a saccharide, a nucleic acid, an organic molecule other than a protein, saccharide, or nucleic acid, a vesicle, or a virus. In various embodiments, a method is provided, wherein the analyte secreted by the biological micro-object is an antibody or, optionally, a glycosylated antibody. In various embodiments, a method is provided, wherein the binding component of the reporter molecule binds to an antibody heavy chain H1 domain. In various embodiments, a method is provided, wherein the binding component of the reporter molecule binds to an antibody light chain (e.g., an epitope that includes one or more amino acid residues of a light chain peptide). In various embodiments, a method is provided, wherein the binding component of the report molecules binds to a lambda light chain (e.g., an epitope that includes one or more amino acid residues of a lambda light chain peptide). In various embodiments, a method is provided, wherein the binding component of the report molecules binds to a kappa light chain (e.g., an epitope that includes one or more amino acid residues of a kappa light chain peptide). In various embodiments, a method is provided, wherein the microfluidic device comprises a plurality of chambers, wherein a biological micro-object is introduced into each of at least two chambers of the plurality, and wherein the remainder of the method is carried out with respect to each of the at least two chambers. In various embodiments, a method is provided, wherein further comprising: comparing a level of secretion for chambers of the at least two chambers of the plurality of chambers. In various embodiments, a method is provided, wherein further comprising: comparing secretion scores of more than one chamber of the plurality of chambers. In various embodiments, a method is provided, wherein introducing the plurality of biological micro-objects into the plurality of chambers of the microfluidic device comprises selecting the biological micro-objects and selectively moving the selected biological micro-objects into the plurality of chambers. In various embodiments, a method is provided, wherein the selection is a positive selection. In various embodiments, a method is provided, wherein the selection is a negative selection. In various embodiments, a method of clonal line development is provided, further comprising: selecting a set of chambers from the plurality of chambers, wherein each chamber of the set has a score indicating that the biological micro-object, or clonal population, contained therein is a top analyte producer; exporting from the microfluidic device one or more biological micro-objects contained within each chamber of the set of selected chambers; expanding the exported one or more biological micro-objects from each chamber of the set of selected chambers in corresponding reaction vessels; and, optionally, determining a level of analyte secreted in each corresponding reaction vessel, thereby determining a level of secretion for each biological micro-object or clonal population. Additional methods are provided in the claims attached hereto. BRIEF DESCRIPTION OF THE DRAWINGSFor a more complete understanding of the principles disclosed herein, and the advantages thereof, reference is now made to the following descriptions taken in conjunction with the accompanying drawings, in which: Figure 1A illustrates an example of a system for use with a microfluidic device and associated control equipment according to some embodiments of the disclosure. Figures 1B and 1C illustrate a microfluidic device according to some embodiments of the disclosure. Figures 2A and 2B illustrate chambers (e.g., isolation chambers) according to some embodiments of the disclosure. Figure 2C illustrates a detailed chamber according to some embodiments of the disclosure. Figures 2D-F illustrate chambers according to some other embodiments of the disclosure. Figure 2G illustrates a microfluidic device according to an embodiment of the disclosure.

Figure 2H illustrates a coated surface of the microfluidic device according to an embodiment of the disclosure. Figure 3A illustrates a detailed chamber according to some other embodiments of the disclosure. Figure 3B illustrates a specific example of a system for use with a microfluidic device and associated control equipment according to some embodiments of the disclosure. Figure 3C illustrates an imaging device according to some embodiments of the disclosure. Figures 4A-4C are graphical representations of an assay according to some embodiments of the disclosure. Figures 5A-5C are graphical illustrations of an assay according to some other embodiments of the disclosure. Figure 6 is a schematic illustration of diffusion characteristics within a chamber of a microfluidic device according to some embodiments of the disclosure. Figures 7A-7B are graphical representations of calculated diffusion rates of molecules according to some embodiments of the disclosure. Figures 8A-8B are graphical representations of calculated and experimentally confirmed diffusion rates of molecules according to some embodiments of the disclosure. Figures 9A-9B are graphical representation of diffusion characteristics within a chamber of a microfluidic device according to some embodiments of the disclosure. Figures 10A-10B are graphical representation of diffusion characteristics within a chamber of a microfluidic device according to some other embodiments of the disclosure. Figures 11A-11B are graphical representation of diffusion characteristics within a chamber of a microfluidic device according to yet other embodiments of the disclosure. Figures 12A-12C are graphical and photographic representations of diffusion characteristics within a chamber of a microfluidic device and an area of interest for assessing levels of secretion of a product from a biological micro-object, according to some embodiments of the disclosure. Figures 13A-13B depict photographic images of a microfluidic device before and after normalization according to some embodiments of the disclosure. Figures 14A-14C are graphical and photographic representations of assay images within a microfluidic device and assay data for an area of interest thereof, according to some embodiments of the disclosure. Figure 15 is a graphical representation of an overlay of median intensity values for a plurality of chambers within a microfluidic device, according to some embodiments of the disclosure.

Figure 16A and 16B are graphical representations of analyte secretion by biological micro-objects disposed within a microfluidic device, according to some embodiments of the disclosure. Figure 17 illustrates steps performed to quantify an amount of analyte secreted by a biological micro-object present in chambers according to some embodiments of the present disclosure. Figure 18 illustrates a sequence of steps performed to calculate an absolute or relative value representing the amount of analyte secreted by a biological micro-object according to some embodiments of the disclosure. Figure 19 illustrates steps performed to assess an absolute or relative value representing the amount of analyte secreted by a clonal population of cells according to some embodiments of the disclosure. Figure 20 is a graphical representation of a titration curve generated according to some embodiments of the disclosure. Figure 21 is a photographic representation of a normalized assay image of a portion of microfluidic device including pen identification and assay scores according to some embodiments of the disclosure. Figure 22 is a photographic and a graphical representation of a course of a culturing and assay sequence according to some embodiments of the disclosure. Figures 23A-23B are graphical representation of assay values for all the chambers of a microfluidic device according to some embodiments of the disclosure. Figure 24 is a graphical representation of correlation between assay values for clonal populations in selected chambers of a microfluidic device and the respective scaled up clonal population according to some embodiments of the disclosure. Figure 25 is a block diagram that illustrates a computer system, in accordance with various embodiments. Figure 26 is a schematic diagram of a system for assessing a quantity of analyte, in accordance with various embodiments Figure 27 is a schematic diagram of a system for assessing a quantity of analyte, in accordance with various embodiments. Figure 28 is a cross-section of a chamber of a micro-fluidic device, in accordance with various embodiments. Figure 29 and 30 are exemplary flowcharts depicting a method for determining a quantity of analyte, in accordance with various embodiments.

Figure 31 are exemplary graphs depicting the relationship between time and fluorescence, in accordance with various embodiments. Figure 32 illustrate chambers with photobleaching, in accordance with various embodiments. Figures 33A-33C illustrates an example cell line development assay for small molecule analytes, in accordance with various embodiments. Figure 34 is a schematic diagram of a system for assessing a quantity of analyte, in accordance with various embodiments. Figure 35 is a cross-section of a chamber of a micro-fluidic device, in accordance with various embodiments. Figure 36 illustrates steps performed to quantify an amount of analyte secreted by a biological micro-object present in chambers according to various embodiments of the present disclosure. Figure 37 illustrates steps performed to quantify an amount of analyte secreted by a biological micro-object present in chambers according to various embodiments of the present disclosure. Figure 38 illustrates steps performed to quantify an amount of analyte secreted by a biological micro-object present in chambers according to various embodiments of the present disclosure. Figure 39 illustrates steps performed to quantify an amount of analyte secreted by a plurality of biological micro-objects present in a plurality of chambers according to various embodiments of the present disclosure. Figures 40A-40G illustrate aspects of a simulation experiment performed to assess improvements for measuring signal from RMSA complex in a hook region of a microfluidic device. Figures 41A-41B illustrate exemplary data comparing a flush assay with an equilibration assay. Figures 42A-42B illustrate exemplary data showing improvement in signal from averaged images taken for an equilibration assay. It is to be understood that the figures are not necessarily drawn to scale, nor are the objects in the figures necessarily drawn to scale in relationship to one another. The figures are depictions that are intended to bring clarity and understanding to various embodiments of apparatuses, systems, and methods disclosed herein. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts. Moreover, it should be appreciated that the drawings are not intended to limit the scope of the present teachings in any way.

DETAILED DESCRIPTION This specification describes exemplary embodiments and applications of the disclosure. The disclosure, however, is not limited to these exemplary embodiments and applications or to the manner in which the exemplary embodiments and applications operate or are described herein. Moreover, the figures may show simplified or partial views, and the dimensions of elements in the figures may be exaggerated or otherwise not in proportion. In addition, as the terms "on," "attached to," "connected to," "coupled to," or similar words are used herein, one element (e.g., a material, a layer, a substrate, etc.) can be "on," "attached to," "connected to," or "coupled to" another element regardless of whether the one element is directly on, attached to, connected to, or coupled to the other element or there are one or more intervening elements between the one element and the other element. Also, unless the context dictates otherwise, directions (e.g., above, below, top, bottom, side, up, down, under, over, upper, lower, horizontal, vertical, "x," "y," "z," etc.), if provided, are relative and provided solely by way of example and for ease of illustration and discussion and not by way of limitation. In addition, where reference is made to a list of elements (e.g., elements a, b, c), such reference is intended to include any one of the listed elements by itself, any combination of less than all of the listed elements, and/or a combination of all of the listed elements. Section divisions in the specification are for ease of review only and do not limit any combination of elements discussed. Where dimensions of microfluidic features are described as having a width or an area, the dimension typically is described relative to an x-axial and/or y-axial dimension, both of which lie within a plane that is parallel to the substrate and/or cover of the microfluidic device. The height of a microfluidic feature may be described relative to a z-axial direction, which is perpendicular to a plane that is parallel to the substrate and/or cover of the microfluidic device. In some instances, a cross sectional area of a microfluidic feature, such as a channel or a passageway, may be in reference to a x-axial/z-axial, a y-axial/z-axial, or an x-axial/y-axial area. As used herein, "substantially" means sufficient to work for the intended purpose. The term "substantially" thus allows for minor, insignificant variations from an absolute or perfect state, dimension, measurement, result, or the like such as would be expected by a person of ordinary skill in the field but that do not appreciably affect overall performance. When used with respect to numerical values or parameters or characteristics that can be expressed as numerical values, "substantially" means within ten percent. The term "ones" means more than one. As used herein, the term "plurality" can be 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. As used herein, the term "disposed" encompasses within its meaning "located." As used herein, a "microfluidic device" or "microfluidic apparatus" is a device that includes one or more discrete microfluidic circuits configured to hold a fluid, each microfluidic circuit comprised of fluidically interconnected circuit elements, including but not limited to region(s), flow path(s), channel(s), chamber(s), and/or pen(s), and at least one port configured to allow the fluid (and, optionally, micro-objects suspended in the fluid) to flow into and/or out of the microfluidic device. Typically, a microfluidic circuit of a microfluidic device will include a flow region, which may include a microfluidic channel, and at least one chamber, and will hold a volume of fluid of less than about 1 mL, e.g., less than about 750, 500, 250, 200, 150, 100, 75, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, or 2 µL. In certain embodiments, the microfluidic circuit holds about 1-2, 1-3, 1-4, 1-5, 2-5, 2-8, 2-10, 2-12, 2-15, 2-20, 5-20, 5-30, 5-40, 5-50, 10-50, 10-75, 10-100, 20-100, 20-150, 20-200, 50-200, 50-250, or 50-300 µL. The microfluidic circuit may be configured to have a first end fluidically connected with a first port (e.g., an inlet) in the microfluidic device and a second end fluidically connected with a second port (e.g., an outlet) in the microfluidic device. As used herein, a "nanofluidic device" or "nanofluidic apparatus" is a type of microfluidic device having a microfluidic circuit that contains at least one circuit element configured to hold a volume of fluid of less than about 1 µL, e.g., less than about 750, 500, 250, 200, 150, 100, 75, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 nL or less. A nanofluidic device may comprise a plurality of circuit elements (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10,000, or more). In certain embodiments, one or more (e.g., all) of the at least one circuit elements is configured to hold a volume of fluid of about 100 pL to 1 nL, 100 pL to 2 nL, 100 pL to 5 nL, 2pL to 2 nL, 250 pL to 5 nL, 250 pL to 10 nL, 500 pL to 5 nL, 500 pL to 10 nL, 500 pL to 15 nL, 7pL to 10 nL, 750 pL to 15 nL, 750 pL to 20 nL, 1 to 10 nL, 1 to 15 nL, 1 to 20 nL, 1 to 25 nL, or 1 to nL. In other embodiments, one or more (e.g., all) of the at least one circuit elements are configured to hold a volume of fluid of about 20 nL to 200nL, 100 to 200 nL, 100 to 300 nL, 100 to 400 nL, 100 to 500 nL, 200 to 300 nL, 200 to 400 nL, 200 to 500 nL, 200 to 600 nL, 200 to 700 nL, 250 to 400 nL, 250 to 500 nL, 250 to 600 nL, or 250 to 750 nL. A microfluidic device or a nanofluidic device may be referred to herein as a "microfluidic chip" or a "chip"; or "nanofluidic chip" or "chip". A "microfluidic channel" or "flow channel" as used herein refers to flow region of a microfluidic device having a length that is significantly longer than both the horizontal and vertical dimensions. For example, the flow channel can be at least 5 times the length of either the horizontal or vertical dimension, e.g., at least 10 times the length, at least 25 times the length, at least 100 times the length, at least 200 times the length, at least 500 times the length, at least 1,000 times the length, at least 5,000 times the length, or longer. In some embodiments, the length of a flow channel is about 100,000 microns to about 500,000 microns, including any value therebetween. In some embodiments, the horizontal dimension is about 100 microns to about 1000 microns (e.g., about 150 to about 500 microns) and the vertical dimension is about 25 microns to about 200 microns, (e.g., from about 40 to about 150 microns). It is noted that a flow channel may have a variety of different spatial configurations in a microfluidic device, and thus is not restricted to a perfectly linear element. For example, a flow channel may be, or include one or more sections having, the following configurations: curve, bend, spiral, incline, decline, fork (e.g., multiple different flow paths), and any combination thereof. In addition, a flow channel may have different cross-sectional areas along its path, widening and constricting to provide a desired fluid flow therein. The flow channel may include valves, and the valves may be of any type known in the art of microfluidics. Examples of microfluidic channels that include valves are disclosed in U.S. Patents 6,408,878 and 9,227,200, each of which is herein incorporated by reference in its entirety. As used herein, the term "obstruction" refers generally to a bump or similar type of structure that is sufficiently large so as to partially (but not completely) impede movement of target micro- objects between two different regions or circuit elements in a microfluidic device. The two different regions/circuit elements can be, for example, the connection region and the isolation region of a microfluidic chamber. As used herein, the term "constriction" refers generally to a narrowing of a width of a circuit element (or an interface between two circuit elements) in a microfluidic device. The constriction can be located, for example, at the interface between the isolation region and the connection region of a microfluidic chamber of the instant disclosure. As used herein, the term "transparent" refers to a material which allows visible light to pass through without substantially altering the light as is passes through. As used herein, "brightfield" illumination and/or image refers to white light illumination of the microfluidic field of view from a broad-spectrum light source, where contrast is formed by absorbance of light by objects in the field of view. As used herein, the term "micro-object" refers generally to any microscopic object that may be isolated and/or manipulated in accordance with the present disclosure. Non-limiting examples of micro-objects include: inanimate micro-objects such as microparticles; microbeads (e.g., polystyrene beads, Luminex™ beads, or the like); magnetic beads; microrods; microwires; quantum dots, and the like; biological micro-objects such as cells; biological organelles; vesicles, or complexes; synthetic vesicles; liposomes (e.g., synthetic or derived from membrane preparations); lipid nanorafts, and the like; or a combination of inanimate micro-objects and biological micro-objects (e.g., microbeads attached to cells, liposome-coated micro-beads, liposome-coated magnetic beads, or the like). Beads may include moieties/molecules covalently or non-covalently attached, such as fluorescent labels, proteins, carbohydrates, antigens, small molecule signaling moieties, or other chemical/biological species capable of use in an assay. Lipid nanorafts have been described, for example, in Ritchie et al. (2009) "Reconstitution of Membrane Proteins in Phospholipid Bilayer Nanodiscs," Methods Enzymol., 464:211-231. As used herein, the term "cell" is used interchangeably with the term "biological cell." Non-limiting examples of biological cells include eukaryotic cells, plant cells, animal cells, such as mammalian cells, reptilian cells, avian cells, fish cells, or the like, prokaryotic cells, bacterial cells, fungal cells, protozoan cells, or the like, cells dissociated from a tissue, such as muscle, cartilage, fat, skin, liver, lung, neural tissue, and the like, immunological cells, such as T cells, B cells, natural killer cells, macrophages, and the like, embryos (e.g., zygotes), oocytes, ova, sperm cells, hybridomas, cultured cells, cells from a cell line, cancer cells, infected cells, transfected and/or transformed cells, reporter cells, and the like. A mammalian cell can be, for example, from a human, a mouse, a rat, a horse, a goat, a sheep, a cow, a primate, or the like. A colony of biological cells is "clonal" if all of the living cells in the colony that are capable of reproducing are daughter cells derived from a single parent cell. In certain embodiments, all the daughter cells in a clonal colony are derived from the single parent cell by no more than 10 divisions. In other embodiments, all the daughter cells in a clonal colony are derived from the single parent cell by no more than 14 divisions. In other embodiments, all the daughter cells in a clonal colony are derived from the single parent cell by no more than 17 divisions. In other embodiments, all the daughter cells in a clonal colony are derived from the single parent cell by no more than 20 divisions. The term "clonal cells" refers to cells of the same clonal colony. As used herein, a "colony" of biological cells refers to 2 or more cells (e.g. about 2 to about 20, about 4 to about 40, about 6 to about 60, about 8 to about 80, about 10 to about 100, about 20 to about 200, about 40 to about 400, about 60 to about 600, about 80 to about 800, about 100 to about 1000, or greater than 1000 cells). As used herein, the term "maintaining (a) cell(s)" refers to providing an environment comprising both fluidic and gaseous components and, optionally a surface, that provides the conditions necessary to keep the cells viable and/or expanding. As used herein, the term "expanding" when referring to cells, refers to increasing in cell number. A "component" of a fluidic medium is any chemical or biochemical molecule present in the medium, including solvent molecules, ions, small molecules, antibiotics, nucleotides and nucleosides, nucleic acids, amino acids, peptides, proteins, sugars, carbohydrates, lipids, fatty acids, cholesterol, metabolites, or the like. As used herein, "capture moiety" is a chemical or biological species, functionality, or motif that provides a recognition site for a micro-object. A selected class of micro-objects may recognize the in situ-generated capture moiety and may bind or have an affinity for the in situ-generated capture moiety. Non-limiting examples include antigens, antibodies, and cell surface binding motifs. As used herein, "flowable polymer" is a polymer monomer or macromer that is soluble or dispersible within a fluidic medium (e.g., a pre-polymer solution). The flowable polymer may be input into a microfluidic flow region and flow with other components of a fluidic medium therein. As used herein, "photoinitiated polymer" refers to a polymer (or a monomeric molecule that can be used to generate the polymer) that upon exposure to light, is capable of crosslinking covalently, forming specific covalent bonds, changing regiochemistry around a rigidified chemical motif, or forming ion pairs which cause a change in physical state, and thereby forming a polymer network. In some instances, a photoinitiated polymer may include a polymer segment bound to one or more chemical moieties capable of crosslinking covalently, forming specific covalent bonds, changing regiochemistry around a rigidified chemical motif, or forming ion pairs which cause a change in physical state. In some instances, a photoinitiated polymer may require a photoactivatable radical initiator to initiate formation of the polymer network (e.g., via polymerization of the polymer). As used herein, "antibody" refers to an immunoglobulin (Ig) and includes both polyclonal and monoclonal antibodies; primatized (e.g., humanized); murine; mouse-human; mouse-primate; and chimeric; and may be an intact molecule, a fragment thereof (such as scFv, Fv, Fd, Fab, Fab' and F(ab)'2 fragments), or multimers or aggregates of intact molecules and/or fragments; and may occur in nature or be produced, e.g., by immunization, synthesis or genetic engineering. An "antibody fragment," as used herein, refers to fragments, derived from or related to an antibody, which bind antigen and which in some embodiments may be derivatized to exhibit structural features that facilitate clearance and uptake, e.g., by the incorporation of galactose residues. This includes, e.g., F(ab), F(ab)'2, scFv, light chain variable region (VL), heavy chain variable region (VH), and combinations thereof. As used herein in reference to a fluidic medium, "diffuse" and "diffusion" refer to thermodynamic movement of a component of the fluidic medium down a concentration gradient. The phrase "flow of a medium" means bulk movement of a fluidic medium primarily due to any mechanism other than diffusion. For example, flow of a medium can involve movement of the fluidic medium from one point to another point due to a pressure differential between the points. Such flow can include a continuous, pulsed, periodic, random, intermittent, or reciprocating flow of the liquid, or any combination thereof. When one fluidic medium flows into another fluidic medium, turbulence and mixing of the media can result. The phrase "substantially no flow" refers to a rate of flow of a fluidic medium that, averaged over time, is less than the rate of diffusion of components of a material (e.g., an analyte of interest) into or within the fluidic medium. The rate of diffusion of components of such a material can depend on, for example, temperature, the size of the components, and the strength of interactions between the components and the fluidic medium. As used herein the term "direct flow" refers to flow in the predominant direction of flow in a flow region of the microfluidic device. In most instances this flow will be laminar flow at a controlled rate determined by the rate of solution moving into the device through an inlet and out of a device through an outlet. Generally, the flow region will be a region, such as a channel, that connects the inlet and the outlet. As used herein the term "secondary flow" refers to fluid flow in a region of a chamber fluidically connected to a flow region, wherein the rate of fluid flow in the region of the chamber experiencing secondary flow is between 100% and 0.1% of the rate of flow in the flow region. In certain embodiments, the rate of fluid flow in the region of the chamber experiencing secondary flow is between 100% and 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, or 5% of the rate of flow in the adjoining flow region. In certain embodiments, the flow region is a channel. In certain embodiments, the secondary flow occurs in a connection region of a sequestration pen. In some instances, secondary flow comprises fluid from the flow region that expands into the chamber at the junction between the flow region and the opening of the chamber to the flow region. In other instances, secondary flow comprises vortices produced in a region of the chamber proximal to the flow region, wherein laminar flow of fluid in the flow region as it flows past and expands into the opening of the chamber is responsible for producing these vortices. The presence of these vortices comprising "secondary flow" can increase a rate of fluid and solute exchange between an adjacent flow region and a chamber in which the "secondary flow" may be disposed (e.g., a chamber comprising a connection region where these vortices may form), with the rate of exchange increasing with an increasing laminar flow rate in the flow region. As used herein, the term "isolation region" refers to a region in a chamber of a microfluidic device that is isolated from both direct flow of fluid in a flow region (e.g., channel) of the microfluidic device and from secondary flow which may be present in another portion of the chamber. Typically, in an isolation region, the rate of movement of solutes by diffusion will be greater than the rate of movement of solutes resulting from fluid flow, and as such diffusion becomes the primary means by which soluble components enter or exit the "isolation region." As used herein in reference to different regions within a microfluidic device, the phrase "fluidically connected" means that, when the different regions are substantially filled with fluid, such as fluidic media, the fluid in each of the regions is connected so as to form a single body of fluid. This does not mean that the fluids (or fluidic media) in the different regions are necessarily identical in composition. Rather, the fluids in different fluidically connected regions of a microfluidic device can have different compositions (e.g., different concentrations of solutes, such as proteins, carbohydrates, ions, or other molecules) which are in flux as solutes move down their respective concentration gradients and/or fluids flow through the microfluidic device. As used herein, a "flow path" refers to one or more fluidically connected circuit elements (e.g. channel(s), region(s), chamber(s) and the like) that define, and are subject to, the trajectory of a flow of medium. A flow path is thus an example of a swept region of a microfluidic device. Other circuit elements (e.g., unswept regions) may be fluidically connected with the circuit elements that comprise the flow path without being subject to the flow of medium in the flow path. As used herein, the term "hook region" refers to any sub-region or area of interest of a microfluidic device that is both unlikely to have cells disposed with in it and is isolated from direct flow in the flow region and/or secondary flow (e.g., flow produced by vortices generated by direct laminar flow in the flow region). As used herein, "isolating a micro-object" confines a micro-object to a defined area within the microfluidic device. As used herein, the term "refresh rate" is the ratio of the volume of the flow region divided by the flow rate. This "refresh rate" represents the amount of time it takes to replace the entire volume of fluid in the flow region. A microfluidic (or nanofluidic) device can comprise "swept" regions and "unswept" regions. As used herein, a "swept" region is comprised of one or more fluidically interconnected circuit elements of a microfluidic circuit, each of which experiences a flow of medium when fluid is flowing through the microfluidic circuit. The circuit elements of a swept region can include, for example, regions, channels, and all or parts of chambers. As used herein, an "unswept" region is comprised of one or more fluidically interconnected circuit element of a microfluidic circuit, each of which experiences substantially no flux of fluid when fluid is flowing through the microfluidic circuit. An unswept region can be fluidically connected to a swept region, provided the fluidic connections are structured to enable diffusion but substantially no flow of media between the swept region and the unswept region. The microfluidic device can thus be structured to substantially isolate an unswept region from a flow of medium in a swept region, while enabling substantially only diffusive fluidic communication between the swept region and the unswept region. For example, a flow channel of a micro-fluidic device is an example of a swept region while an isolation region (described in further detail below) of a microfluidic device is an example of an unswept region. The capability of biological micro-objects (e.g., biological cells) to produce specific biological materials (e.g., proteins, such as antibodies) can be assayed in such a microfluidic device. In a specific embodiment of an assay, sample material comprising biological micro-objects (e.g., cells) to be assayed for production of an analyte of interest can be loaded into a swept region of the microfluidic device. Ones of the biological micro-objects (e.g., mammalian cells, such as human cells) can be selected for particular characteristics and disposed in unswept regions. The remaining sample material can then be flowed out of the swept region and an assay material flowed into the swept region. Because the selected biological micro-objects are in unswept regions, the selected biological micro-objects are not substantially affected by the flowing out of the remaining sample material or the flowing in of the assay material. The selected biological micro-objects can be allowed to produce the analyte of interest, which can diffuse from the unswept regions into the swept region, where the analyte of interest can react with the assay material to produce localized detectable reactions, each of which can be correlated to a particular unswept region. Any unswept region associated with a detected reaction can be analyzed to assess which, if any, of the biological micro-objects in the unswept region are sufficient producers of the analyte of interest. Microfluidic devices and systems for operating and observing such devices. Figure 1A illustrates an example of a microfluidic device 100 and a system 150 which can be used for maintaining, isolating, assaying or culturing biological micro-objects. A perspective view of the microfluidic device 100 is shown having a partial cut-away of its cover 110 to provide a partial view into the microfluidic device 100. The microfluidic device 100 generally comprises a microfluidic circuit 120 comprising a flow path 106 through which a fluidic medium 180 can flow, optionally carrying one or more micro-objects (not shown) into and/or through the microfluidic circuit 120. Although a single microfluidic circuit 120 is illustrated in Figure 1A, suitable microfluidic devices can include a plurality (e.g., 2 or 3) of such microfluidic circuits. Regardless, the microfluidic device 100 can be configured to be a nanofluidic device. As illustrated in Figure 1A, the microfluidic circuit 120 may include a plurality of microfluidic chambers 124, 126, 128, and 130, where each chambers may have one or more openings in fluidic communication with flow path 106. In some embodiments of the device of Figure 1A, the chambers may have only a single opening in fluidic communication with the flow path 106. As discussed further below, the microfluidic chambers comprise various features and structures that have been optimized for retaining micro-objects in the microfluidic device, such as microfluidic device 100, even when a medium 180 is flowing through the flow path 106. Before turning to the foregoing, however, a brief description of microfluidic device 100 and system 150 is provided. As generally illustrated in Figure 1A, the microfluidic circuit 120 is defined by an enclosure 102. Although the enclosure 102 can be physically structured in different configurations, in the example shown in Figure 1A the enclosure 102 is depicted as comprising a support structure 104 (e.g., a base), a microfluidic circuit structure 108, and a cover 110. The support structure 104, microfluidic circuit structure 108, and cover 110 can be attached to each other. For example, the microfluidic circuit structure 108 can be disposed on an inner surface 109 of the support structure 104, and the cover 110 can be disposed over the microfluidic circuit structure 108. Together with the support structure 104 and cover 110, the microfluidic circuit structure 108 can define the elements of the microfluidic circuit 120. The support structure 104 can be at the bottom and the cover 110 at the top of the microfluidic circuit 120 as illustrated in Figure 1A. Alternatively, the support structure 104 and the cover 110 can be configured in other orientations. For example, the support structure 104 can be at the top and the cover 110 at the bottom of the microfluidic circuit 120. Regardless, there can be one or more ports 107 each comprising a passage into or out of the enclosure 102. Examples of a passage include a valve, a gate, a pass-through hole, or the like. As illustrated, port 107 is a pass-through hole created by a gap in the microfluidic circuit structure 108. However, the port 107 can be situated in other components of the enclosure 102, such as the cover 110. Only one port 107 is illustrated in Figure 1A but the microfluidic circuit 120 can have two or more ports 107. For example, there can be a first port 107 that functions as an inlet for fluid entering the microfluidic circuit 120, and there can be a second port 107 that functions as an outlet for fluid exiting the microfluidic circuit 120. Whether a port 107 function as an inlet or an outlet can depend upon the direction that fluid flows through flow path 106. The support structure 104 can comprise one or more electrodes (not shown) and a substrate or a plurality of interconnected substrates. For example, the support structure 104 can comprise one or more semiconductor substrates, each of which is electrically connected to an electrode (e.g., all or a subset of the semiconductor substrates can be electrically connected to a single electrode). The support structure 104 can further comprise a printed circuit board assembly ("PCBA"). For example, the semiconductor substrate(s) can be mounted on a PCBA. The microfluidic circuit structure 108 can define circuit elements of the microfluidic circuit 120. Such circuit elements can comprise spaces or regions that can be fluidly interconnected when microfluidic circuit 120 is filled with fluid, such as flow regions (which may include or be one or more flow channels), chambers, pens, traps, and the like. In the microfluidic circuit 120 illustrated in Figure 1A, the microfluidic circuit structure 108 comprises a frame 114 and a microfluidic circuit material 116. The frame 114 can partially or completely enclose the microfluidic circuit material 116. The frame 114 can be, for example, a relatively rigid structure substantially surrounding the microfluidic circuit material 116. For example, the frame 114 can comprise a metal material. The microfluidic circuit material 116 can be patterned with cavities or the like to define circuit elements and interconnections of the microfluidic circuit 120. The microfluidic circuit material 116 can comprise a flexible material, such as a flexible polymer (e.g. rubber, plastic, elastomer, silicone, polydimethylsiloxane ("PDMS"), or the like), which can be gas permeable. Other examples of materials that can compose microfluidic circuit material 116 include molded glass, an etchable material such as silicone (e.g. photo-patternable silicone or "PPS"), photo-resist (e.g., SU8), or the like. In some embodiments, such materials—and thus the microfluidic circuit material 116—can be rigid and/or substantially impermeable to gas. Regardless, microfluidic circuit material 1can be disposed on the support structure 104 and inside the frame 114. The cover 110 can be an integral part of the frame 114 and/or the microfluidic circuit material 116. Alternatively, the cover 110 can be a structurally distinct element, as illustrated in Figure 1A. The cover 110 can comprise the same or different materials than the frame 114 and/or the microfluidic circuit material 116. Similarly, the support structure 104 can be a separate structure from the frame 114 or microfluidic circuit material 116 as illustrated, or an integral part of the frame 114 or microfluidic circuit material 116. Likewise, the frame 114 and microfluidic circuit material 116 can be separate structures as shown in Figure 1A or integral portions of the same structure. In some embodiments, the cover 110 can comprise a rigid material. The rigid material may be glass or a material with similar properties. In some embodiments, the cover 110 can comprise a deformable material. The deformable material can be a polymer, such as PDMS. In some embodiments, the cover 110 can comprise both rigid and deformable materials. For example, one or more portions of cover 110 (e.g., one or more portions positioned over chambers 124, 126, 128, 130) can comprise a deformable material that interfaces with rigid materials of the cover 110. In some embodiments, the cover 110 can further include one or more electrodes. The one or more electrodes can comprise a conductive oxide, such as indium-tin-oxide (ITO), which may be coated on glass or a similarly insulating material. Alternatively, the one or more electrodes can be flexible electrodes, such as single-walled nanotubes, multi-walled nanotubes, nanowires, clusters of electrically conductive nanoparticles, or combinations thereof, embedded in a deformable material, such as a polymer (e.g., PDMS). Flexible electrodes that can be used in microfluidic devices have been described, for example, in U.S. 2012/0325665 (Chiou et al.), the contents of which are incorporated herein by reference. In some embodiments, the cover 110 can be modified (e.g., by conditioning all or part of a surface that faces inward toward the microfluidic circuit 120) to support cell adhesion, viability and/or growth. The modification may include a coating of a synthetic or natural polymer. In some embodiments, the cover 110 and/or the support structure 104 can be transparent to light. The cover 110 may also include at least one material that is gas permeable (e.g., PDMS or PPS). Figure 1A also shows a system 150 for operating and controlling microfluidic devices, such as microfluidic device 100. System 150 includes an electrical power source 192, an imaging device 194 (incorporated within imaging module 164, where device 194 is not illustrated in Figure 1A, per se), and a tilting device 190 (part of tilting module 166, where device 190 is not illustrated in Figure 1A). The electrical power source 192 can provide electric power to the microfluidic device 100 and/or tilting device 190, providing biasing voltages or currents as needed. The electrical power source 192 can, for example, comprise one or more alternating current (AC) and/or direct current (DC) voltage or current sources. The imaging device 194 (part of imaging module 164, discussed below) can comprise a device, such as a digital camera, for capturing images inside microfluidic circuit 120. In some instances, the imaging device 194 further comprises a detector having a fast frame rate and/or high sensitivity (e.g. for low light applications). The imaging device 194 can also include a mechanism for directing stimulating radiation and/or light beams into the microfluidic circuit 120 and collecting radiation and/or light beams reflected or emitted from the microfluidic circuit 120 (or micro-objects contained therein). The emitted light beams may be in the visible spectrum and may, e.g., include fluorescent emissions. The reflected light beams may include reflected emissions originating from an LED or a wide spectrum lamp, such as a mercury lamp (e.g. a high pressure mercury lamp) or a Xenon arc lamp. As discussed with respect to Figure 3B, the imaging device 194 may further include a microscope (or an optical train), which may or may not include an eyepiece. System 150 further comprises a tilting device 190 (part of tilting module 166, discussed below) configured to rotate a microfluidic device 100 about one or more axes of rotation. In some embodiments, the tilting device 190 is configured to support and/or hold the enclosure 1comprising the microfluidic circuit 120 about at least one axis such that the microfluidic device 1(and thus the microfluidic circuit 120) can be held in a level orientation (i.e. at 0° relative to x- and y-axes), a vertical orientation (i.e. at 90° relative to the x-axis and/or the y-axis), or any orientation therebetween. The orientation of the microfluidic device 100 (and the microfluidic circuit 120) relative to an axis is referred to herein as the "tilt" of the microfluidic device 100 (and the microfluidic circuit 120). For example, the tilting device 190 can tilt the microfluidic device 100 at 0.1°, 0.2°, 0.3°, 0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1°, 2°, 3°, 4°, 5°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°, 55°, 60°, 65°, 70°, 75°, 80°, 90° relative to the x-axis or any degree therebetween. The level orientation (and thus the x- and y-axes) is defined as normal to a vertical axis defined by the force of gravity. The tilting device can also tilt the microfluidic device 100 (and the microfluidic circuit 120) to any degree greater than 90° relative to the x-axis and/or y-axis, or tilt the microfluidic device 1(and the microfluidic circuit 120) 180° relative to the x-axis or the y-axis in order to fully invert the microfluidic device 100 (and the microfluidic circuit 120). Similarly, in some embodiments, the tilting device 190 tilts the microfluidic device 100 (and the microfluidic circuit 120) about an axis of rotation defined by flow path 106 or some other portion of microfluidic circuit 120. In some instances, the microfluidic device 100 is tilted into a vertical orientation such that the flow path 106 is positioned above or below one or more chambers. The term "above" as used herein denotes that the flow path 106 is positioned higher than the one or more chambers on a vertical axis defined by the force of gravity (i.e. an object in a chamber above a flow path 106 would have a higher gravitational potential energy than an object in the flow path). The term "below" as used herein denotes that the flow path 106 is positioned lower than the one or more chambers on a vertical axis defined by the force of gravity (i.e. an object in a chamber below a flow path 106 would have a lower gravitational potential energy than an object in the flow path). In some instances, the tilting device 190 tilts the microfluidic device 100 about an axis that is parallel to the flow path 106. Moreover, the microfluidic device 100 can be tilted to an angle of less than 90° such that the flow path 106 is located above or below one or more chambers without being located directly above or below the chambers. In other instances, the tilting device 190 tilts the microfluidic device 100 about an axis perpendicular to the flow path 106. In still other instances, the tilting device 190 tilts the microfluidic device 100 about an axis that is neither parallel nor perpendicular to the flow path 106. System 150 can further include a media source 178. The media source 178 (e.g., a container, reservoir, or the like) can comprise multiple sections or containers, each for holding a different fluidic medium 180. Thus, the media source 178 can be a device that is outside of and separate from the microfluidic device 100, as illustrated in Figure 1A. Alternatively, the media source 178 can be located in whole or in part inside the enclosure 102 of the microfluidic device 100. For example, the media source 178 can comprise reservoirs that are part of the microfluidic device 100. Figure 1A also illustrates simplified block diagram depictions of examples of control and monitoring equipment 152 that constitute part of system 150 and can be utilized in conjunction with a microfluidic device 100. As shown, examples of such control and monitoring equipment 152 include a master controller 154 comprising a media module 160 for controlling the media source 178, a motive module 162 for controlling movement and/or selection of micro-objects (not shown) and/or medium (e.g., droplets of medium) in the microfluidic circuit 120, an imaging module 164 for controlling an imaging device 194 (e.g., a camera, microscope, light source or any combination thereof) for capturing images (e.g., digital images), and a tilting module 166 for controlling a tilting device 190. The control equipment 152 can also include other modules 168 for controlling, monitoring, or performing other functions with respect to the microfluidic device 100. As shown, the equipment 152 can further include a display device 170 and an input/output device 172. The master controller 154 can comprise a control module 156 and a digital memory 158. The control module 156 can comprise, for example, a digital processor configured to operate in accordance with machine executable instructions (e.g., software, firmware, source code, or the like) stored as non-transitory data or signals in the memory 158. Alternatively, or in addition, the control module 156 can comprise hardwired digital circuitry and/or analog circuitry. The media module 160, motive module 162, imaging module 164, tilting module 166, and/or other modules 168 can be similarly configured. Thus, functions, processes acts, actions, or steps of a process discussed herein as being performed with respect to the microfluidic device 100 or any other microfluidic apparatus can be performed by any one or more of the master controller 154, media module 160, motive module 162, imaging module 164, tilting module 166, and/or other modules 168 configured as discussed above. Similarly, the master controller 154, media module 160, motive module 162, imaging module 164, tilting module 166, and/or other modules 168 may be communicatively coupled to transmit and receive data used in any function, process, act, action or step discussed herein. The media module 160 controls the media source 178. For example, the media module 160 can control the media source 178 to input a selected fluidic medium 180 into the enclosure 102 (e.g., through an inlet port 107). The media module 160 can also control removal of media from the enclosure 102 (e.g., through an outlet port (not shown)). One or more media can thus be selectively input into and removed from the microfluidic circuit 120. The media module 160 can also control the flow of fluidic medium 180 in the flow path 106 inside the microfluidic circuit 120. For example, in some embodiments media module 160 stops the flow of media 180 in the flow path 106 and through the enclosure 102 prior to the tilting module 166 causing the tilting device 190 to tilt the microfluidic device 100 to a desired angle of incline. The motive module 162 can be configured to control selection, trapping, and movement of micro-objects (not shown) in the microfluidic circuit 120. As discussed below with respect to Figures 1B and 1C, the enclosure 102 can comprise a dielectrophoresis (DEP), optoelectronic tweezers (OET) and/or opto-electrowetting (OEW) configuration (not shown in Figure 1A), and the motive module 162 can control the activation of electrodes and/or transistors (e.g., phototransistors) to select and move micro-objects (not shown) and/or droplets of medium (not shown) in the flow path 106 and/or chambers 124, 126, 128, 130. The imaging module 164 can control the imaging device 194. For example, the imaging module 164 can receive and process image data from the imaging device 194. Image data from the imaging device 194 can comprise any type of information captured by the imaging device 194 (e.g., the presence or absence of micro-objects, droplets of medium, accumulation of label, such as fluorescent label, etc.). Using the information captured by the imaging device 194, the imaging module 164 can further calculate the position of objects (e.g., micro-objects, droplets of medium) and/or the rate of motion of such objects within the microfluidic device 100. The tilting module 166 can control the tilting motions of tilting device 190. Alternatively, or in addition, the tilting module 166 can control the tilting rate and timing to optimize transfer of micro-objects to the one or more chambers via gravitational forces. The tilting module 166 is communicatively coupled with the imaging module 164 to receive data describing the motion of micro-objects and/or droplets of medium in the microfluidic circuit 120. Using this data, the tilting module 166 may adjust the tilt of the microfluidic circuit 120 in order to adjust the rate at which micro-objects and/or droplets of medium move in the microfluidic circuit 120. The tilting module 166 may also use this data to iteratively adjust the position of a micro-object and/or droplet of medium in the microfluidic circuit 120. In the example shown in Figure 1A, the microfluidic circuit 120 is illustrated as comprising a microfluidic channel 122 and chambers 124, 126, 128, 130. Each chamber comprises an opening to channel 122, but otherwise is enclosed such that the chambers can substantially isolate micro-objects inside the pen from fluidic medium 180 and/or micro-objects in the flow path 106 of channel 122 or in other chambers. The walls of the chamber extend from the inner surface 109 of the base to the inside surface of the cover 110 to provide enclosure. The opening of the chamber to the microfluidic channel 122 is oriented at an angle to the flow 106 of fluidic medium 180 such that flow 106 is not directed into the chambers. The flow may be tangential or orthogonal to the plane of the opening of the chamber. In some instances, chambers 124, 126, 128, 130 are configured to physically corral one or more micro-objects within the microfluidic circuit 120. Chambers in accordance with the present disclosure can comprise various shapes, surfaces and features that are optimized for use with DEP, OET, OEW, fluid flow, and/or gravitational forces, as will be discussed and shown in detail below. The microfluidic circuit 120 may comprise any number of microfluidic chambers. Although five chambers are shown, microfluidic circuit 120 may have fewer or more chambers. As shown, microfluidic chambers 124, 126, 128, and 130 of microfluidic circuit 120 each comprise differing features and shapes which may provide one or more benefits useful for maintaining, isolating, assaying or culturing biological micro-objects. In some embodiments, the microfluidic circuit 1comprises a plurality of identical microfluidic chambers. In the embodiment illustrated in Figure 1A, a single channel 122 and flow path 106 is shown. However, other embodiments may contain multiple channels 122, each configured to comprise a flow path 106. The microfluidic circuit 120 further comprises an inlet valve or port 107 in fluid communication with the flow path 106 and fluidic medium 180, whereby fluidic medium 180 can access channel 122 via the inlet port 107. In some instances, the flow path 106 comprises a single path. In some instances, the single path is arranged in a zigzag pattern whereby the flow path 1travels across the microfluidic device 100 two or more times in alternating directions. The flow in the flow path 106 may proceed from inlet to outlet or may be reversed and proceed from outlet to inlet. In some instances, microfluidic circuit 120 comprises a plurality of parallel channels 122 and flow paths 106, wherein the fluidic medium 180 within each flow path 106 flows in the same direction. In some instances, the fluidic medium within each flow path 106 flows in at least one of a forward or reverse direction. In some instances, a plurality of chambers is configured (e.g., relative to a channel 122) such that the chambers can be loaded with target micro-objects in parallel. In some embodiments, microfluidic circuit 120 further comprises one or more micro-object traps 132. The traps 132 are generally formed in a wall forming the boundary of a channel 122, and may be positioned opposite an opening of one or more of the microfluidic chambers 124, 126, 128, 130. In some embodiments, the traps 132 are configured to receive or capture a single micro-object from the flow path 106. In some embodiments, the traps 132 are configured to receive or capture a plurality of micro-objects from the flow path 106. In some instances, the traps 132 comprise a volume approximately equal to the volume of a single target micro-object. The traps 132 may further comprise an opening which is configured to assist the flow of targeted micro-objects into the traps 132. In some instances, the traps 132 comprise an opening having a height and width that is approximately equal to the dimensions of a single target micro- object, whereby larger micro-objects are prevented from entering into the micro-object trap. The traps 132 may further comprise other features configured to assist in retention of targeted micro-objects within the trap 132. In some instances, the trap 132 is aligned with and situated on the opposite side of a channel 122 relative to the opening of a microfluidic chamber, such that upon tilting the microfluidic device 100 about an axis parallel to the microfluidic channel 122, the trapped micro-object exits the trap 132 at a trajectory that causes the micro-object to fall into the opening of the chamber. In some instances, the trap 132 comprises a side passage 134 that is smaller than the target micro-object in order to facilitate flow through the trap 132 and thereby increase the likelihood of capturing a micro-object in the trap 132.

In some embodiments, dielectrophoretic (DEP) forces are applied across the fluidic medium 180 (e.g., in the flow path and/or in the chambers) via one or more electrodes (not shown) to manipulate, transport, separate and sort micro-objects located therein. For example, in some embodiments, DEP forces are applied to one or more portions of microfluidic circuit 120 in order to transfer a single micro-object from the flow path 106 into a desired microfluidic chamber. In some embodiments, DEP forces are used to prevent a micro-object within a chamber (e.g., chamber 124, 126, 128, or 130) from being displaced therefrom. Further, in some embodiments, DEP forces are used to selectively remove a micro-object from a chamber that was previously collected in accordance with the embodiments of the current disclosure. In some embodiments, the DEP forces comprise optoelectronic tweezer (OET) forces. In other embodiments, optoelectrowetting (OEW) forces are applied to one or more positions in the support structure 104 (and/or the cover 110) of the microfluidic device 100 (e.g., positions helping to define the flow path and/or the chambers) via one or more electrodes (not shown) to manipulate, transport, separate and sort droplets located in the microfluidic circuit 120. For example, in some embodiments, OEW forces are applied to one or more positions in the support structure 104 (and/or the cover 110) in order to transfer a single droplet from the flow path 106 into a desired microfluidic chamber. In some embodiments, OEW forces are used to prevent a droplet within a chamber (e.g., chamber 124, 126, 128, or 130) from being displaced therefrom. Further, in some embodiments, OEW forces are used to selectively remove a droplet from a chamber that was previously collected in accordance with the embodiments of the current disclosure. In some embodiments, DEP and/or OEW forces are combined with other forces, such as flow and/or gravitational force, so as to manipulate, transport, separate and sort micro-objects and/or droplets within the microfluidic circuit 120. For example, the enclosure 102 can be tilted (e.g., by tilting device 190) to position the flow path 106 and micro-objects located therein above the microfluidic chambers, and the force of gravity can transport the micro-objects and/or droplets into the chambers. In some embodiments, the DEP and/or OEW forces can be applied prior to the other forces. In other embodiments, the DEP and/or OEW forces can be applied after the other forces. In still other instances, the DEP and/or OEW forces can be applied at the same time as the other forces or in an alternating manner with the other forces. Figures 1B, 1C, and 2A-2H illustrates various embodiments of microfluidic devices that can be used in the practice of the embodiments of the present disclosure. Figure 1B depicts an embodiment in which the microfluidic device 200 is configured as an optically-actuated electrokinetic device. A variety of optically-actuated electrokinetic devices are known in the art, including devices having an optoelectronic tweezer (OET) configuration and devices having an opto-electrowetting (OEW) configuration. Examples of suitable OET configurations are illustrated in the following U.S. patent documents, each of which is incorporated herein by reference in its entirety: U.S. Patent No. RE 44,711 (Wu et al.) (originally issued as U.S. Patent No. 7,612,355); and U.S. Patent No. 7,956,339 (Ohta et al.). Examples of OEW configurations are illustrated in U.S. Patent No. 6,958,1(Chiou et al.) and U.S. Patent Application Publication No. 2012/0024708 (Chiou et al.), both of which are incorporated by reference herein in their entirety. Yet another example of an optically-actuated electrokinetic device includes a combined OET/OEW configuration, examples of which are shown in U.S. Patent Publication Nos. 20150306598 (Khandros et al.) and 20150306599 (Khandros et al.) and their corresponding PCT Publications WO2015/164846 and WO2015/164847, all of which are incorporated herein by reference in their entirety. Examples of microfluidic devices having chambers in which biological micro-objects can be placed, cultured, and/or monitored have been described, for example, in US 2014/01168(application no. 14/060,117, filed October 22, 2013), US 2015/0151298 (application no. 14/520,568, filed October 22, 2014), and US 2015/0165436 (application no. 14/521,447, filed October 22, 2014), each of which is incorporated herein by reference in its entirety. US application nos. 14/520,568 and 14/521,447 also describe exemplary methods of analyzing secretions of cells cultured in a microfluidic device. Each of the foregoing applications further describes microfluidic devices configured to produce dielectrophoretic (DEP) forces, such as optoelectronic tweezers (OET) or configured to provide opto-electro wetting (OEW). For example, the optoelectronic tweezers device illustrated in Figure 2 of US 2014/0116881 is an example of a device that can be utilized in embodiments of the present disclosure to select and move an individual biological micro-object or a group of biological micro-objects. Microfluidic device motive configurations. As described above, the control and monitoring equipment of the system can comprise a motive module for selecting and moving objects, such as micro-objects or droplets, in the microfluidic circuit of a microfluidic device. The microfluidic device can have a variety of motive configurations, depending upon the type of object being moved and other considerations. For example, a dielectrophoresis (DEP) configuration can be utilized to select and move micro-objects in the microfluidic circuit. Thus, the support structure 104 and/or cover 110 of the microfluidic device 100 can comprise a DEP configuration for selectively inducing DEP forces on micro-objects in a fluidic medium 180 in the microfluidic circuit 120 and thereby select, capture, and/or move individual micro-objects or groups of micro-objects. Alternatively, the support structure 104 and/or cover 110 of the microfluidic device 100 can comprise an electrowetting (EW) configuration for selectively inducing EW forces on droplets in a fluidic medium 180 in the microfluidic circuit 120 and thereby select, capture, and/or move individual droplets or groups of droplets. One example of a microfluidic device 200 comprising a DEP configuration is illustrated in Figures 1B and 1C. While for purposes of simplicity Figures 1B and 1C show a side cross-sectional view and a top cross-sectional view, respectively, of a portion of an enclosure 102 of the microfluidic device 200 having a region/chamber 202, it should be understood that the region/chamber 202 may be part of a fluidic circuit element having a more detailed structure, such as a growth chamber, a chamber, a flow region, or a flow channel. Furthermore, the microfluidic device 200 may include other fluidic circuit elements. For example, the microfluidic device 200 can include a plurality of growth chambers or chambers and/or one or more flow regions or flow channels, such as those described herein with respect to microfluidic device 100. A DEP configuration may be incorporated into any such fluidic circuit elements of the microfluidic device 200, or select portions thereof. It should be further appreciated that any of the above or below described microfluidic device components and system components may be incorporated in and/or used in combination with the microfluidic device 200. For example, system 150 including control and monitoring equipment 152, described above, may be used with microfluidic device 200, including one or more of the media module 160, motive module 162, imaging module 164, tilting module 166, and other modules 168. As seen in Figure 1B, the microfluidic device 200 includes a support structure 104 having a bottom electrode 204 and an electrode activation substrate 206 overlying the bottom electrode 204, and a cover 110 having a top electrode 210, with the top electrode 210 spaced apart from the bottom electrode 204. The top electrode 210 and the electrode activation substrate 206 define opposing surfaces of the region/chamber 202. A medium 180 contained in the region/chamber 202 thus provides a resistive connection between the top electrode 210 and the electrode activation substrate 206. A power source 212 configured to be connected to the bottom electrode 204 and the top electrode 210 and create a biasing voltage between the electrodes, as required for the generation of DEP forces in the region/chamber 202, is also shown. The power source 212 can be, for example, an alternating current (AC) power source. In certain embodiments, the microfluidic device 200 illustrated in Figures 1B and 1C can have an optically-actuated DEP configuration. Accordingly, changing patterns of light 218 from the light source 216, which may be controlled by the motive module 162, can selectively activate and deactivate changing patterns of DEP electrodes at regions 214 of the inner surface 208 of the electrode activation substrate 206. (Hereinafter the regions 214 of a microfluidic device having a DEP configuration are referred to as "DEP electrode regions.") As illustrated in Figure 1C, a light pattern 218 directed onto the inner surface 208 of the electrode activation substrate 206 can illuminate select DEP electrode regions 214a (shown in white) in a pattern, such as a square. The non-illuminated DEP electrode regions 214 (cross-hatched) are hereinafter referred to as "dark" DEP electrode regions 214. The relative electrical impedance through the DEP electrode activation substrate 206 (i.e., from the bottom electrode 204 up to the inner surface 208 of the electrode activation substrate 206 which interfaces with the medium 180 in the flow region 106) is greater than the relative electrical impedance through the medium 180 in the region/chamber 202 (i.e., from the inner surface 208 of the electrode activation substrate 206 to the top electrode 210 of the cover 110) at each dark DEP electrode region 214. An illuminated DEP electrode region 214a, however, exhibits a reduced relative impedance through the electrode activation substrate 206 that is less than the relative impedance through the medium 180 in the region/chamber 202 at each illuminated DEP electrode region 214a. With the power source 212 activated, the foregoing DEP configuration creates an electric field gradient in the fluidic medium 180 between illuminated DEP electrode regions 214a and adjacent dark DEP electrode regions 214, which in turn creates local DEP forces that attract or repel nearby micro-objects (not shown) in the fluidic medium 180. DEP electrodes that attract or repel micro-objects in the fluidic medium 180 can thus be selectively activated and deactivated at many different such DEP electrode regions 214 at the inner surface 208 of the region/chamber 202 by changing light patterns 218 projected from a light source 216 into the microfluidic device 200. Whether the DEP forces attract or repel nearby micro-objects can depend on such parameters as the frequency of the power source 212 and the dielectric properties of the medium 180 and/or micro- objects (not shown). The square pattern 220 of illuminated DEP electrode regions 214a illustrated in Figure 1C is an example only. Any pattern of the DEP electrode regions 214 can be illuminated (and thereby activated) by the pattern of light 218 projected into the microfluidic device 200, and the pattern of illuminated/activated DEP electrode regions 214 can be repeatedly changed by changing or moving the light pattern 218. In some embodiments, the electrode activation substrate 206 can comprise or consist of a photoconductive material. In such embodiments, the inner surface 208 of the electrode activation substrate 206 can be featureless. For example, the electrode activation substrate 206 can comprise or consist of a layer of hydrogenated amorphous silicon (a-Si:H). The a-Si:H can comprise, for example, about 8% to 40% hydrogen (calculated as 100 * the number of hydrogen atoms / the total number of hydrogen and silicon atoms). The layer of a-Si:H can have a thickness of about 500 nm to about 2.0 m. In such embodiments, the DEP electrode regions 214 can be created anywhere and in any pattern on the inner surface 208 of the electrode activation substrate 206, in accordance with the light pattern 218. The number and pattern of the DEP electrode regions 214 thus need not be fixed, but can correspond to the light pattern 218. Examples of microfluidic devices having a DEP configuration comprising a photoconductive layer such as discussed above have been described, for example, in U.S. Patent No. RE 44,711 (Wu et al.) (originally issued as U.S. Patent No. 7,612,355), the entire contents of which are incorporated herein by reference. In other embodiments, the electrode activation substrate 206 can comprise a substrate comprising a plurality of doped layers, electrically insulating layers (or regions), and electrically conductive layers that form semiconductor integrated circuits, such as is known in semiconductor fields. For example, the electrode activation substrate 206 can comprise a plurality of phototransistors, including, for example, lateral bipolar phototransistors, each phototransistor corresponding to a DEP electrode region 214. Alternatively, the electrode activation substrate 2can comprise electrodes (e.g., conductive metal electrodes) controlled by phototransistor switches, with each such electrode corresponding to a DEP electrode region 214. The electrode activation substrate 206 can include a pattern of such phototransistors or phototransistor-controlled electrodes. The pattern, for example, can be an array of substantially square phototransistors or phototransistor- controlled electrodes arranged in rows and columns, such as shown in Fig. 2B. Alternatively, the pattern can be an array of substantially hexagonal phototransistors or phototransistor-controlled electrodes that form a hexagonal lattice. Regardless of the pattern, electric circuit elements can form electrical connections between the DEP electrode regions 214 at the inner surface 208 of the electrode activation substrate 206 and the bottom electrode 210, and those electrical connections (i.e., phototransistors or electrodes) can be selectively activated and deactivated by the light pattern 218. When not activated, each electrical connection can have high impedance such that the relative impedance through the electrode activation substrate 206 (i.e., from the bottom electrode 204 to the inner surface 208 of the electrode activation substrate 206 which interfaces with the medium 180 in the region/chamber 202) is greater than the relative impedance through the medium 180 (i.e., from the inner surface 208 of the electrode activation substrate 206 to the top electrode 210 of the cover 110) at the corresponding DEP electrode region 214. When activated by light in the light pattern 218, however, the relative impedance through the electrode activation substrate 206 is less than the relative impedance through the medium 180 at each illuminated DEP electrode region 214, thereby activating the DEP electrode at the corresponding DEP electrode region 214 as discussed above. DEP electrodes that attract or repel micro-objects (not shown) in the medium 180 can thus be selectively activated and deactivated at many different DEP electrode regions 214 at the inner surface 208 of the electrode activation substrate 206 in the region/chamber 202 in a manner determined by the light pattern 218.

Examples of microfluidic devices having electrode activation substrates that comprise phototransistors have been described, for example, in U.S. Patent No. 7,956,339 (Ohta et al.) (see, e.g., device 300 illustrated in Figures 21 and 22, and descriptions thereof), the entire contents of which are incorporated herein by reference. Examples of microfluidic devices having electrode activation substrates that comprise electrodes controlled by phototransistor switches have been described, for example, in U.S. Patent Publication No. 2014/0124370 (Short et al.) (see, e.g., devices 200, 400, 500, 600, and 900 illustrated throughout the drawings, and descriptions thereof), the entire contents of which are incorporated herein by reference. In some embodiments of a DEP configured microfluidic device, the top electrode 210 is part of a first wall (or cover 110) of the enclosure 102, and the electrode activation substrate 206 and bottom electrode 204 are part of a second wall (or support structure 104) of the enclosure 102. The region/chamber 202 can be between the first wall and the second wall. In other embodiments, the electrode 210 is part of the second wall (or support structure 104) and one or both of the electrode activation substrate 206 and/or the electrode 210 are part of the first wall (or cover 110). Moreover, the light source 216 can alternatively be used to illuminate the enclosure 102 from below. With the microfluidic device 200 of Figures 1B-1C having a DEP configuration, the motive module 162 can select a micro-object (not shown) in the medium 180 in the region/chamber 202 by projecting a light pattern 218 into the microfluidic device 200 to activate a first set of one or more DEP electrodes at DEP electrode regions 214a of the inner surface 208 of the electrode activation substrate 206 in a pattern (e.g., square pattern 220) that surrounds and captures the micro-object. The motive module 162 can then move the in situ-generated captured micro-object by moving the light pattern 218 relative to the microfluidic device 200 to activate a second set of one or more DEP electrodes at DEP electrode regions 214. Alternatively, the microfluidic device 200 can be moved relative to the light pattern 218. In other embodiments, the microfluidic device 200 can have a DEP configuration that does not rely upon light activation of DEP electrodes at the inner surface 208 of the electrode activation substrate 206. For example, the electrode activation substrate 206 can comprise selectively addressable and energizable electrodes positioned opposite to a surface including at least one electrode (e.g., cover 110). Switches (e.g., transistor switches in a semiconductor substrate) may be selectively opened and closed to activate or inactivate DEP electrodes at DEP electrode regions 214, thereby creating a net DEP force on a micro-object (not shown) in region/chamber 202 in the vicinity of the activated DEP electrodes. Depending on such characteristics as the frequency of the power source 212 and the dielectric properties of the medium (not shown) and/or micro-objects in the region/chamber 202, the DEP force can attract or repel a nearby micro-object. By selectively activating and deactivating a set of DEP electrodes (e.g., at a set of DEP electrodes regions 214 that forms a square pattern 220), one or more micro-objects in region/chamber 202 can be trapped and moved within the region/chamber 202. The motive module 162 in Figure 1A can control such switches and thus activate and deactivate individual ones of the DEP electrodes to select, trap, and move particular micro-objects (not shown) around the region/chamber 202. Microfluidic devices having a DEP configuration that includes selectively addressable and energizable electrodes are known in the art and have been described, for example, in U.S. Patent Nos. 6,294,063 (Becker et al.) and 6,942,776 (Medoro), the entire contents of which are incorporated herein by reference. As yet another example, the microfluidic device 200 can have an electrowetting (EW) configuration, which can be in place of the DEP configuration or can be located in a portion of the microfluidic device 200 that is separate from the portion which has the DEP configuration. The EW configuration can be an opto-electrowetting configuration or an electrowetting on dielectric (EWOD) configuration, both of which are known in the art. In some EW configurations, the support structure 104 has an electrode activation substrate 206 sandwiched between a dielectric layer (not shown) and the bottom electrode 204. The dielectric layer can comprise a hydrophobic material and/or can be coated with a hydrophobic material, as described below. For microfluidic devices 200 that have an EW configuration, the inner surface 208 of the support structure 104 is the inner surface of the dielectric layer or its hydrophobic coating. The dielectric layer (not shown) can comprise one or more oxide layers, and can have a thickness of about 50 nm to about 250 nm (e.g., about 125 nm to about 175 nm). In certain embodiments, the dielectric layer may comprise a layer of oxide, such as a metal oxide (e.g., aluminum oxide or hafnium oxide). In certain embodiments, the dielectric layer can comprise a dielectric material other than a metal oxide, such as silicon oxide or a nitride. Regardless of the exact composition and thickness, the dielectric layer can have an impedance of about 10 kOhms to about kOhms. In some embodiments, the surface of the dielectric layer that faces inward toward region/chamber 202 is coated with a hydrophobic material. The hydrophobic material can comprise, for example, fluorinated carbon molecules. Examples of fluorinated carbon molecules include perfluoro-polymers such as polytetrafluoroethylene (e.g., TEFLON®) or poly(2,3-difluoromethylenyl-perfluorotetrahydrofuran) (e.g., CYTOP™). Molecules that make up the hydrophobic material can be covalently bonded to the surface of the dielectric layer. For example, molecules of the hydrophobic material can be covalently bound to the surface of the dielectric layer by means of a linker such as a siloxane group, a phosphonic acid group, or a thiol group. Thus, in some embodiments, the hydrophobic material can comprise alkyl-terminated siloxane, alkyl- termination phosphonic acid, or alkyl-terminated thiol. The alkyl group can be long-chain hydrocarbons (e.g., having a chain of at least 10 carbons, or at least 16, 18, 20, 22, or more carbons). Alternatively, fluorinated (or perfluorinated) carbon chains can be used in place of the alkyl groups. Thus, for example, the hydrophobic material can comprise fluoroalkyl-terminated siloxane, fluoroalkyl-terminated phosphonic acid, or fluoroalkyl-terminated thiol. In some embodiments, the hydrophobic coating has a thickness of about 10 nm to about 50 nm. In other embodiments, the hydrophobic coating has a thickness of less than 10 nm (e.g., less than 5 nm, or about 1.5 to 3.0 nm). In some embodiments, the cover 110 of a microfluidic device 200 having an electrowetting configuration is coated with a hydrophobic material (not shown) as well. The hydrophobic material can be the same hydrophobic material used to coat the dielectric layer of the support structure 104, and the hydrophobic coating can have a thickness that is substantially the same as the thickness of the hydrophobic coating on the dielectric layer of the support structure 104. Moreover, the cover 110 can comprise an electrode activation substrate 206 sandwiched between a dielectric layer and the top electrode 210, in the manner of the support structure 104. The electrode activation substrate 206 and the dielectric layer of the cover 110 can have the same composition and/or dimensions as the electrode activation substrate 206 and the dielectric layer of the support structure 104. Thus, the microfluidic device 200 can have two electrowetting surfaces. In some embodiments, the electrode activation substrate 206 can comprise a photoconductive material, such as described above. Accordingly, in certain embodiments, the electrode activation substrate 206 can comprise or consist of a layer of hydrogenated amorphous silicon (a-Si:H). The a- Si:H can comprise, for example, about 8% to 40% hydrogen (calculated as 100 * the number of hydrogen atoms / the total number of hydrogen and silicon atoms). The layer of a-Si:H can have a thickness of about 500 nm to about 2.0 m. Alternatively, the electrode activation substrate 206 can comprise electrodes (e.g., conductive metal electrodes) controlled by phototransistor switches, as described above. Microfluidic devices having an opto-electrowetting configuration are known in the art and/or can be constructed with electrode activation substrates known in the art. For example, U.S. Patent No. 6,958,132 (Chiou et al.), the entire contents of which are incorporated herein by reference, discloses opto-electrowetting configurations having a photoconductive material such as a-Si:H, while U.S. Patent Publication No. 2014/0124370 (Short et al.), referenced above, discloses electrode activation substrates having electrodes controlled by phototransistor switches. The microfluidic device 200 thus can have an opto-electrowetting configuration, and light patterns 218 can be used to activate photoconductive EW regions or photoresponsive EW electrodes in the electrode activation substrate 206. Such activated EW regions or EW electrodes of the electrode activation substrate 206 can generate an electrowetting force at the inner surface 208 of the support structure 104 (i.e., the inner surface of the overlaying dielectric layer or its hydrophobic coating). By changing the light patterns 218 (or moving microfluidic device 200 relative to the light source 216) incident on the electrode activation substrate 206, droplets (e.g., containing an aqueous medium, solution, or solvent) contacting the inner surface 208 of the support structure 104 can be moved through an immiscible fluid (e.g., an oil medium) present in the region/chamber 202. In other embodiments, microfluidic devices 200 can have an EWOD configuration, and the electrode activation substrate 206 can comprise selectively addressable and energizable electrodes that do not rely upon light for activation. The electrode activation substrate 206 thus can include a pattern of such electrowetting (EW) electrodes. The pattern, for example, can be an array of substantially square EW electrodes arranged in rows and columns, such as shown in Fig. 2B. Alternatively, the pattern can be an array of substantially hexagonal EW electrodes that form a hexagonal lattice. Regardless of the pattern, the EW electrodes can be selectively activated (or deactivated) by electrical switches (e.g., transistor switches in a semiconductor substrate). By selectively activating and deactivating EW electrodes in the electrode activation substrate 206, droplets (not shown) contacting the inner surface 208 of the overlaying dielectric layer or its hydrophobic coating can be moved within the region/chamber 202. The motive module 162 in Figure 1A can control such switches and thus activate and deactivate individual EW electrodes to select and move particular droplets around region/chamber 202. Microfluidic devices having a EWOD configuration with selectively addressable and energizable electrodes are known in the art and have been described, for example, in U.S. Patent No. 8,685,344 (Sundarsan et al.), the entire contents of which are incorporated herein by reference. Regardless of the configuration of the microfluidic device 200, a power source 212 can be used to provide a potential (e.g., an AC voltage potential) that powers the electrical circuits of the microfluidic device 200. The power source 212 can be the same as, or a component of, the power source 192 referenced in Fig. 1. Power source 212 can be configured to provide an AC voltage and/or current to the top electrode 210 and the bottom electrode 204. For an AC voltage, the power source 212 can provide a frequency range and an average or peak power (e.g., voltage or current) range sufficient to generate net DEP forces (or electrowetting forces) strong enough to trap and move individual micro-objects (not shown) in the region/chamber 202, as discussed above, and/or to change the wetting properties of the inner surface 208 of the support structure 104 (i.e., the dielectric layer and/or the hydrophobic coating on the dielectric layer) in the region/chamber 202, as also discussed above. Such frequency ranges and average or peak power ranges are known in the art. See, e.g., US Patent No. 6,958,132 (Chiou et al.), US Patent No. RE44,711 (Wu et al.) (originally issued as US Patent No. 7,612,355), and US Patent Application Publication Nos. US2014/0124370 (Short et al.), US2015/0306598 (Khandros et al.), and US2015/0306599 (Khandros et al.). Chambers. Non-limiting examples of generic chambers 224, 226, and 228 are shown within the microfluidic device 230 depicted in Figures 2A-2C. Each chamber 224, 226, and 228 can comprise an isolation structure 232 defining an isolation region 240 and a connection region 236 fluidically connecting the isolation region 240 to a channel 122. The connection region 236 can comprise a proximal opening 234 to the microfluidic channel 122 and a distal opening 238 to the isolation region 240. The connection region 236 can be configured so that the maximum penetration depth of a flow of a fluidic medium (not shown) flowing from the microfluidic channel 122 into the chamber 224, 226, 228 does not extend into the isolation region 240. In some embodiments, streamlines from the flow in the microfluidic channel do not enter the isolation region. Thus, due to the connection region 236, a micro-object (not shown) or other material (not shown) disposed in an isolation region 240 of a chamber 224, 226, 228 can thus be isolated from, and not substantially affected by, a flow of medium 180 in the microfluidic channel 122. The chambers 224, 226, and 228 of Figures 2A-2C each have a single opening which opens directly to the microfluidic channel 122. The opening of the chamber may open laterally from the microfluidic channel 122. The electrode activation substrate 206 underlays both the microfluidic channel 122 and the chambers 224, 226, and 228. The upper surface of the electrode activation substrate 206 within the enclosure of a chamber, forming the floor of the chamber, is disposed at the same level or substantially the same level of the upper surface the of electrode activation substrate 206 within the microfluidic channel 122 (or flow region if a channel is not present), forming the floor of the flow channel (or flow region, respectively) of the microfluidic device. The electrode activation substrate 206 may be featureless or may have an irregular or patterned surface that varies from its highest elevation to its lowest depression by less than about 3 microns, 2.5 microns, 2 microns, 1.microns, 1 micron, 0.9 microns, 0.5 microns, 0.4 microns, 0.2 microns, 0.1 microns or less. The variation of elevation in the upper surface of the substrate across both the microfluidic channel 1(or flow region) and chambers may be less than about 3%, 2%, 1%. 0.9%, 0.8%, 0.5%, 0.3% or 0.1% of the height of the walls of the chamber or walls of the microfluidic device. While described in detail for the microfluidic device 200, this also applies to any of the microfluidic devices 100, 230, 250, 280, 290, 300, 400, 500, 900, 1000, 1100, 1200 described herein. The microfluidic channel 122 can thus be an example of a swept region, and the isolation regions 240 of the chambers 224, 226, 228 can be examples of unswept regions. Chambers like 224, 226, 228 may have isolation regions wherein each isolation region has only one opening, which opens to the connection region of the sequestration pen. Fluidic media exchange in and out of the isolation region so configured can be limited to occurring substantially only by diffusion. As noted, the microfluidic channel 122 and chambers 224, 226, 228 can be configured to contain one or more fluidic media 180. In the example shown in Figures 2A-2B, the ports 222 are connected to the microfluidic channel 122 and allow a fluidic medium 180 to be introduced into or removed from the microfluidic device 230. Prior to introduction of the fluidic medium 180, the microfluidic device may be primed with a gas such as carbon dioxide gas. Once the microfluidic device 230 contains the fluidic medium 180, the flow 242 of fluidic medium 180 in the microfluidic channel 122 can be selectively generated and stopped. For example, as shown, the ports 222 can be disposed at different locations (e.g., opposite ends) of the microfluidic channel 122, and a flow 242 of medium can be created from one port 222 functioning as an inlet to another port 222 functioning as an outlet. Figure 2C illustrates a detailed view of an example of a chamber 224 according to the present disclosure. Examples of micro-objects 246 are also shown. As is known, a flow 242 of fluidic medium 180 in a microfluidic channel 122 past a proximal opening 234 of chamber 224 can cause a secondary flow 244 of the medium 180 into and/or out of the chamber 224. To isolate micro-objects 246 in the isolation region 240 of a chamber 224 from the secondary flow 244, the length L con of the connection region 236 of the chamber 224 (i.e., from the proximal opening 234 to the distal opening 238) should be greater than the penetration depth D p of the secondary flow 244 into the connection region 236. The penetration depth D p depends upon a number of factors, including the shape of the microfluidic channel 122, which may be defined by a width W con of the connection region 236 at the proximal opening 234; a width W ch of the microfluidic channel 122 at the proximal opening 234; a height H ch of the channel 122 at the proximal opening 234; and the width of the distal opening 238 of the connection region 236. Of these factors, the width W con of the connection region 236 at the proximal opening 234 and the height H ch of the channel 1at the proximal opening 234 tend to be the most significant. In addition, the penetration depth D p can be influenced by the velocity of the fluidic medium 180 in the channel 122 and the viscosity of fluidic medium 180. However, these factors (i.e., velocity and viscosity) can vary widely without dramatic changes in penetration depth D p. For example, for a microfluidic chip 230 having a width W con of the connection region 236 at the proximal opening 234 of about 50 microns, a height H ch of the channel 122 at the proximal opening 122 of about 40 microns, and a width W ch of the microfluidic channel 122 at the proximal opening 122 of about 100 microns to about 150 microns, the penetration depth D p of the secondary flow 244 ranges from less than 1.0 times W con (i.e., less than 50 microns) at a flow rate of 0.1 microliters/sec to about 2.0 times W con (i.e., about 100 microns) at a flow rate of microliters/sec, which represents an increase in D p of only about 2.5-fold over a 200-fold increase in the velocity of the fluidic medium 180.Accordingly, for each chamber 224, a maximal velocity V max for the flow 242 of fluidic medium 180 in channel 122 can be identified that ensures that the penetration depth D p of the secondary flow 244 does not exceed the length L con of the connection region 236. As long as the rate of the flow 242 of fluidic medium 180 in the microfluidic channel 1does not exceed the maximum velocity V max, the resulting secondary flow 244 can be wholly contained within the connection region 236 and does not enter the isolation region 240. The flow 242 of medium 180 in the microfluidic channel 122 will thus not draw micro-objects 246 out of the isolation region 240. Rather, micro-objects 246 located in the isolation region 240 will stay in the isolation region 240 regardless of the flow 242 of fluidic medium 180 in the microfluidic channel 122. Moreover, as long as the rate of flow 242 of medium 180 in the microfluidic channel 122 does not exceed V max, the flow 242 of fluidic medium 180 in the microfluidic channel 122 will not move miscellaneous particles (e.g., microparticles and/or nanoparticles) from the microfluidic channel 122 into the isolation region 240 of a chamber 224. Having the length L con of the connection region 236 be greater than the maximum penetration depth D p of the secondary flow 244 can thus prevent contamination of one chamber 224 with miscellaneous particles from the microfluidic channel 122 or another chamber (e.g., chambers 226, 228 in Fig. 2D). Because the microfluidic channel 122 and the connection regions 236 of the chambers 224, 226, 228 can be affected by the flow 242 of medium 180 in the microfluidic channel 122, the microfluidic channel 122 and connection regions 236 can be deemed swept (or flow) regions of the microfluidic device 230. The isolation regions 240 of the chambers 224, 226, 228, on the other hand, can be deemed unswept (or non-flow) regions. For example, components (not shown) in a first fluidic medium 180 in the microfluidic channel 122 can mix with a second fluidic medium 248 in the isolation region 240 substantially only by diffusion of components of the first medium 180 from the microfluidic channel 122 through the connection region 236 and into the second fluidic medium 2in the isolation region 240. Similarly, components (not shown) of the second medium 248 in the isolation region 240 can mix with the first medium 180 in the microfluidic channel 122 substantially only by diffusion of components of the second medium 248 from the isolation region 240 through the connection region 236 and into the first medium 180 in the microfluidic channel 122. In some embodiments, the extent of fluidic medium exchange between the isolation region of a chamber and the flow region by diffusion is greater than about 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or greater than about 99% of fluidic exchange. The first medium 180 can be the same medium or a different medium than the second medium 248. Moreover, the first medium 180 and the second medium 248 can start out being the same, then become different (e.g., through conditioning of the second medium 248 by one or more cells in the isolation region 240, or by changing the medium 1flowing through the microfluidic channel 122). In some embodiments, the dimensions of the microfluidic channel 122 and chambers 224, 226, 228 can be oriented as follows with respect to the vector of the flow 242 of fluidic medium 1in the microfluidic channel 122: the microfluidic channel width W ch (or cross-sectional area of the microfluidic channel 122) can be substantially perpendicular to the flow 242 of medium 180; the width W con (or cross-sectional area) of the connection region 236 at opening 234 can be substantially parallel to the flow 242 of medium 180 in the microfluidic channel 122; and/or the length L con of the connection region can be substantially perpendicular to the flow 242 of medium 180 in the microfluidic channel 122. The foregoing are examples only, and the relative position of the microfluidic channel 122 and chambers 224, 226, 228 can be in other orientations with respect to each other. As illustrated in Figure 2C, the width W con of the connection region 236 can be uniform from the proximal opening 234 to the distal opening 238. The width W con of the connection region 236 at the distal opening 238 can thus be any of the values identified herein for the width W con of the connection region 236 at the proximal opening 234. Alternatively, the width W con of the connection region 236 at the distal opening 238 can be larger than the width W con of the connection region 236 at the proximal opening 234. As illustrated in Figure 2C, the width of the isolation region 240 at the distal opening 238 can be substantially the same as the width W con of the connection region 236 at the proximal opening 234. The width of the isolation region 240 at the distal opening 238 can thus be any of the values identified herein for the width W con of the connection region 236 at the proximal opening 234. Alternatively, the width of the isolation region 240 at the distal opening 238 can be larger or smaller than the width W con of the connection region 236 at the proximal opening 234. Moreover, the distal opening 2may be smaller than the proximal opening 234 and the width W con of the connection region 236 may be narrowed between the proximal opening 234 and distal opening 238. For example, the connection region 236 may be narrowed between the proximal opening and the distal opening, using a variety of different geometries (e.g. chamfering the connection region, beveling the connection region). Further, any part or subpart of the connection region 236 may be narrowed (e.g. a portion of the connection region adjacent to the proximal opening 234). Figures 2D-2F depict another exemplary embodiment of a microfluidic device 250 containing a microfluidic circuit 262 and flow channels 264, which are variations of the respective microfluidic device 100, circuit 132 and channel 134 of Figure 1A. The microfluidic device 250 also has a plurality of chambers 266 that are additional variations of the above-described chambers 124, 126, 128, 130, 224, 226 or 228. In particular, it should be appreciated that the chambers 266 of device 2shown in Figures 2D-2F can replace any of the above-described chambers 124, 126, 128, 130, 224, 226 or 228 in devices 100, 200, 230, 250, 280, 290, 300 400, 500, 900, 1000, 1100, 1200. Likewise, the microfluidic device 250 is another variant of the microfluidic device 100, and may also have the same or a different DEP configuration as the above-described microfluidic device 100, 200, 230, 250, 280, 290, 300, 400, 500, 900, 1000, 1100, 1200 as well as any of the other microfluidic system components described herein. The microfluidic device 250 of Figures 2D-2Fcomprises a support structure (not visible in Figures 2D-2F, but can be the same or generally similar to the support structure 104 of device 1depicted in Figure 1A), a microfluidic circuit structure 256, and a cover (not visible in Figures 2D-2F, but can be the same or generally similar to the cover 122 of device 100 depicted in Figure 1A). The microfluidic circuit structure 256 includes a frame 252 and microfluidic circuit material 260, which can be the same as or generally similar to the frame 114 and microfluidic circuit material 116 of device 100 shown in Figure 1A. As shown in Figure 2D, the microfluidic circuit 262 defined by the microfluidic circuit material 260 can comprise multiple channels 264 (two are shown but there can be more) to which multiple chambers 266 are fluidically connected. Each chamber 266 can comprise an isolation structure 272, an isolation region 270 within the isolation structure 272, and a connection region 268. From a proximal opening 274 at the microfluidic channel 264 to a distal opening 276 at the isolation structure 272, the connection region 268 fluidically connects the microfluidic channel 264 to the isolation region 270. Generally, in accordance with the above discussion of Figures 2B and 2C, a flow 278 of a first fluidic medium 2in a channel 264 can create secondary flows 282 of the first medium 254 from the microfluidic channel 264 into and/or out of the respective connection regions 268 of the chambers 266. As illustrated in Figure 2E, the connection region 268 of each chamber 266 generally includes the area extending between the proximal opening 274 to a channel 264 and the distal opening 276 to an isolation structure 272. The length L con of the connection region 268 can be greater than the maximum penetration depth D p of secondary flow 282, in which case the secondary flow 2will extend into the connection region 268 without being redirected toward the isolation region 2(as shown in Figure 2D). Alternatively, at illustrated in Figure 2F, the connection region 268 can have a length L con that is less than the maximum penetration depth D p, in which case the secondary flow 282 will extend through the connection region 268 and be redirected toward the isolation region 270. In this latter situation, the sum of lengths L c1 and L c2 of connection region 268 is greater than the maximum penetration depth D p, so that secondary flow 282 will not extend into isolation region 270. Whether length L con of connection region 268 is greater than the penetration depth D p, or the sum of lengths L c1 and L c2 of connection region 268 is greater than the penetration depth D p, a flow 278 of a first medium 254 in channel 264 that does not exceed a maximum velocity V max will produce a secondary flow having a penetration depth D p, and micro-objects (not shown but can be the same or generally similar to the micro-objects 246 shown in Figure 2C) in the isolation region 270 of a chamber 266 will not be drawn out of the isolation region 270 by a flow 278 of first medium 254 in channel 264. Nor will the flow 278 in channel 264 draw miscellaneous materials (not shown) from channel 264 into the isolation region 270 of a chamber 266. As such, diffusion is the only mechanism by which components in a first medium 254 in the microfluidic channel 264 can move from the microfluidic channel 264 into a second medium 258 in an isolation region 270 of a chamber 266. Likewise, diffusion is the only mechanism by which components in a second medium 258 in an isolation region 270 of a chamber 266 can move from the isolation region 270 to a first medium 2in the microfluidic channel 264. The first medium 254 can be the same medium as the second medium 258, or the first medium 254 can be a different medium than the second medium 258. Alternatively, the first medium 254 and the second medium 258 can start out being the same, then become different, e.g., through conditioning of the second medium by one or more cells in the isolation region 270, or by changing the medium flowing through the microfluidic channel 264. As illustrated in Figure 2E, the width W ch of the microfluidic channels 264 (i.e., taken transverse to the direction of a fluid medium flow through the microfluidic channel indicated by arrows 278 in Figure 2D) in the microfluidic channel 264 can be substantially perpendicular to a width W con1 of the proximal opening 274 and thus substantially parallel to a width W con2 of the distal opening 276. The width W con1 of the proximal opening 274 and the width W con2 of the distal opening 276, however, need not be substantially perpendicular to each other. For example, an angle between an axis (not shown) on which the width W con1 of the proximal opening 274 is oriented and another axis on which the width W con2 of the distal opening 276 is oriented can be other than perpendicular and thus other than 90°. Examples of alternatively oriented angles include angles of: about 30° to about 90°, about 45° to about 90°, about 60° to about 90°, or the like. FIG. 3A depicts another exemplary embodiment of a microfluidic device 300 containing microfluidic circuit structure 308, which includes a channel 322 and chamber 324, which has features and properties like any of the chambers described herein for microfluidic devices 100, 200, 230, 250, 280, 290, 300 400, 500, 900, 1000, 1100, 1200and any other microfluidic devices described herein. The exemplary microfluidic devices of FIG. 3A includes a microfluidic channel 322, having a width W ch, as described herein, and containing a flow 310 of first fluidic medium 302 and one or more chambers 324 (only one illustrated in FIG. 3). The chambers 324 each have a length L s, a connection region 336, and an isolation region 340, where the isolation region 340 contains a second fluidic medium 304. The connection region 336 has a proximal opening 334, having a width W con1, which opens to the microfluidic channel 322, and a distal opening 338, having a width W con2, which opens to the isolation region 340. The width W con1 may or may not be the same as W con2, as described herein. The walls of each chamber 324 may be formed of microfluidic circuit material 316, which may further form the connection region walls 330. A connection region wall 330 can correspond to a structure that is laterally positioned with respect to the proximal opening 334 and at least partially extends into the enclosed portion of the chamber 324. In some embodiments, the length L con of the connection region 336 is at least partially defined by length L wall of the connection region wall 330. The connection region wall 330 may have a length L wall, selected to be more than the penetration depth D p of the secondary flow 344. Thus, the secondary flow 344 can be wholly contained within the connection region without extending into the isolation region 340. The connection region wall 330 may define a hook region 352, which is a sub-region of the isolation region 340 of the chamber 324. Although the term "hook region" is used to refer to this sub-region of the isolation region 340, a "hook region" can be more broadly construed as any region of the microfluidic chamber that is isolated from direct and/or secondary flow and is unlikely to or does not have cells in it. Since the connection region wall 330 extends into the inner cavity of the chamber, the connection region wall 330 can act as a physical barrier to shield hook region 352 from secondary flow 344, with selection of the length of L wall, contributing to the extent of the hook region. In some embodiments, the longer the length L wall of the connection region wall 330, the more sheltered the hook region 352. In chambers configured like those of FIGS. 2A-2G and 3A, the isolation region may have a shape and size of any type, and may be selected to regulate diffusion of nutrients, reagents, and/or media into the chamber to reach to a far wall of the chamber, e.g., opposite the proximal opening of the connection region to the flow region (or microfluidic channel). The size and shape of the isolation region may further be selected to regulate diffusion of waste products and/or secreted products of a biological micro-object out from the isolation region to the flow region via the proximal opening of the connection region of the chamber. In general, the shape of the isolation region is not critical to the ability of the chamber to isolate micro-objects from direct flow in the flow region. In some other embodiments of chambers, the isolation region may have more than one opening fluidically connecting the isolation region with the flow region of the microfluidic device. However, for an isolation region having a number of n openings fluidically connecting the isolation region to the flow region (or two or more flow regions), n-1 openings can be valved. When the n-valved openings are closed, the isolation region has only one effective opening, and exchange of materials into/out of the isolation region occurs only by diffusion.

Microfluidic circuit element dimensions.Various dimensions and/or features of the sequestration pens and the microfluidic channels to which the sequestration pens open, as described herein, may be selected to limit introduction of contaminants or unwanted micro-objects into the isolation region of a sequestration pen from the flow region/microfluidic channel; limit the exchange of components in the fluidic medium from the channel or from the isolation region to substantially only diffusive exchange; facilitate the transfer of micro-objects into and/or out of the sequestration pens; and/or facilitate growth or expansion of the biological cells. Microfluidic channels and sequestration pens, for any of the embodiments described herein, may have any suitable combination of dimensions, may be selected by one of skill from the teachings of this disclosure. For any of the microfluidic devices described herein, a microfluidic channel may have a uniform cross sectional height along its length that is a substantially uniform cross sectional height, and may be any cross sectional height as described herein. At any point along the microfluidic channel, the substantially uniform cross sectional height of the channel, the upper surface of which is defined by the inner surface of the cover and the lower surface of which is defined by the inner surface of the base, may be substantially the same as the cross sectional height at any other point along the channel, e.g., having a cross sectional height that is no more than about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2% or about 1% or less, different from the cross-sectional height of any other location within the channel. Additionally, the chamber(s), e.g., sequestration pen(s), of the microfluidic devices described herein, may be disposed substantially in a coplanar orientation relative to the microfluidic channel into which the chamber(s) open. That is, the enclosed volume of the chamber(s) is formed by an upper surface that is defined by the inner surface of the cover, a lower surface defined by the inner surface of the base, and walls defined by the microfluidic circuit material. Therefore, the lower surface of the chamber(s) may be coplanar to the lower surface of the microfluidic channel, e.g., substantially coplanar. The upper surface of the chamber may be may be coplanar to the upper surface of the microfluidic channel, e.g., substantially coplanar. Accordingly the chamber(s) may have a cross-sectional height, which may have any values as described herein, that is the same as the channel, e.g., substantially the same, and the chamber(s) and microfluidic channel(s) within the microfluidic device may have a substantially uniform cross sectional height throughout the flow region of the microfluidic device, and may be substantially coplanar throughout the microfluidic device. Coplanarity of the lower surfaces of the chamber(s) and the microfluidic channel(s) can offer distinct advantage with repositioning micro-objects within the microfluidic device using DEP or magnetic force. Penning and unpenning of micro-objects, and in particular selective penning/ selective unpenning, can be greatly facilitated when the lower surfaces of the chamber(s) and the microfluidic channel to which the chamber(s) open have a coplanar orientation. The proximal opening of the connection region of a sequestration pen may have a width (e.g., W con or W con1) that is at least as large as the largest dimension of a micro-object (e.g., a biological cell, which may be a plant cell, such as a plant protoplast) for which the sequestration pen is intended. In some embodiments, the proximal opening has a width (e.g., W con or W con1) of about 20 microns, about microns, about 50 microns, about 60 microns, about 75 microns, about 100 microns, about 1microns, about 200 microns, or about 300 microns. The foregoing are examples only, and the width (e.g., W con or W con1) of a proximal opening can be selected to be a value between any of the values listed above (e.g., about 20-200 microns, about 20-150 microns, about 20-100 microns, about 20-75 microns, about 20-60 microns, about 50-300 microns, about 50-200 microns, about 50-150 microns, about 50-100 microns, about 50-75 microns, about 75-150 microns, about 75-100 microns, about 100-300 microns, about 100-200 microns, or about 200-300 microns). In some embodiments, the connection region of the sequestration pen may have a length (e.g., L con) from the proximal opening to the distal opening to the isolation region of the sequestration pen that is at least 0.5 times, at least 0.6 times, at least 0.7 times, at least 0.8 times, at least 0.9 times, at least 1.0 times, at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.75 times, at least 2.0 times, at least 2.25. times, at least 2.5 times, at least 2.75 times, at least 3.times, at least 3.5 times, at least 4.0 times, at least 4.5 times, at least 5.0 times, at least 6.0 times, at least 7.0 times, at least 8.0 times, at least 9.0 times, or at least 10.0 times the width (e.g., W con or W con1) of the proximal opening. Thus, for example, the proximal opening of the connection region of a sequestration pen may have a width (e.g., W con or W con1) from about 20 microns to about 200 microns (e.g., about 50 microns to about 150 microns), and the connection region may have a length L con that is at least 1.0 times (e.g., at least 1.5 times, or at least 2.0 times) the width of the proximal opening. As another example, the proximal opening of the connection region of a sequestration pen may have a width (e.g., W con or W con1) from about 20 microns to about 100 microns (e.g., about 20 microns to about microns), and the connection region may have a length L con that is at least 1.0 times (e.g., at least 1.5 times, or at least 2.0 times) the width of the proximal opening. The microfluidic channel of a microfluidic device to which a sequestration pen opens may have specified size (e.g., width or height). In some embodiments, the height (e.g., H ch) of the microfluidic channel at a proximal opening to the connection region of a sequestration pen can be within any of the following ranges: 20-100 microns, 20-90 microns, 20-80 microns, 20-70 microns, 20-60 microns, 20-microns, 30-100 microns, 30-90 microns, 30-80 microns, 30-70 microns, 30-60 microns, 30-microns, 40-100 microns, 40-90 microns, 40-80 microns, 40-70 microns, 40-60 microns, or 40-50 microns. The foregoing are examples only, and the height (e.g., H ch) of the microfluidic channel (e.g., 122) can be selected to be between any of the values listed above. Moreover, the height (e.g., H ch) of the microfluidic channel 122 can be selected to be any of these heights in regions of the microfluidic channel other than at a proximal opening of a sequestration pen. The width (e.g., W ch) of the microfluidic channel at the proximal opening to the connection region of a sequestration pen can be within any of the following ranges: about 20-500 microns, 20-4microns, 20-300 microns, 20-200 microns, 20-150 microns, 20-100 microns, 20-80 microns, 20-microns, 30-400 microns, 30-300 microns, 30-200 microns, 30-150 microns, 30-100 microns, 30-microns, 30-60 microns, 40-300 microns, 40-200 microns, 40-150 microns, 40-100 microns, 40-microns, 40-60 microns, 50-1000 microns, 50-500 microns, 50-400 microns, 50-300 microns, 50-250 microns, 50-200 microns, 50-150 microns, 50-100 microns, 50-80 microns, 60-300 microns, 60-2microns, 60-150 microns, 60-100 microns, 60-80 microns, 70-500 microns, 70-400 microns, 70-3microns, 70-250 microns, 70-200 microns, 70-150 microns, 70-100 microns, 80-100 microns, 90-4microns, 90-300 microns, 90-250 microns, 90-200 microns, 90-150 microns, 100-300 microns, 100-250 microns, 100-200 microns, 100-150 microns, 100-120 microns, 200-800 microns, 200-700 microns, or 200-600 microns. The foregoing are examples only, and the width (e.g., W ch) of the microfluidic channel can be a value selected to be between any of the values listed above. Moreover, the width (e.g., W ch) of the microfluidic channel can be selected to be in any of these widths in regions of the microfluidic channel other than at a proximal opening of a sequestration pen. In some embodiments, the width W ch of the microfluidic channel at the proximal opening to the connection region of the sequestration pen (e.g., taken transverse to the direction of bulk flow of fluid through the channel) can be substantially perpendicular to a width (e.g., W con or W con1) of the proximal opening. A cross-sectional area of the microfluidic channel at a proximal opening to the connection region of a sequestration pen can be about 500-50,000 square microns, 500-40,000 square microns, 500-30,000 square microns, 500-25,000 square microns, 500-20,000 square microns, 500-15,000 square microns, 500-10,000 square microns, 500-7,500 square microns, 500-5,000 square microns, 1,000-25,000 square microns, 1,000-20,000 square microns, 1,000-15,000 square microns, 1,000-10,000 square microns, 1,000-7,500 square microns, 1,000-5,000 square microns, 2,000-20,000 square microns, 2,000-15,000 square microns, 2,000-10,000 square microns, 2,000-7,500 square microns, 2,000-6,000 square microns, 3,000-20,000 square microns, 3,000-15,000 square microns, 3,000-10,000 square microns, 3,000-7,500 square microns, or 3,000 to 6,000 square microns. The foregoing are examples only, and the cross-sectional area of the microfluidic channel at the proximal opening can be selected to be between any of the values listed above. In various embodiments, and the cross-sectional area of the microfluidic channel at regions of the microfluidic channel other than at the proximal opening can also be selected to be between any of the values listed above. In some embodiments, the cross-sectional area is selected to be a substantially uniform value for the entire length of the microfluidic channel. In some embodiments, the microfluidic chip is configured such that the proximal opening (e.g., 234 or 334) of the connection region of a sequestration pen may have a width (e.g., W con or W con1) from about 20 microns to about 200 microns (e.g., about 50 microns to about 150 microns), the connection region may have a length L con (e.g., 236 or 336) that is at least 1.0 times (e.g., at least 1.5 times, or at least 2.0 times) the width of the proximal opening, and the microfluidic channel may have a height (e.g., H ch) at the proximal opening of about 30 microns to about 60 microns. As another example, the proximal opening (e.g., 234 or 334) of the connection region of a sequestration pen may have a width (e.g., W con or W con1) from about 20 microns to about 100 microns (e.g., about 20 microns to about microns), the connection region may have a length L con (e.g., 236 or 336) that is at least 1.0 times (e.g., at least 1.5 times, or at least 2.0 times) the width of the proximal opening, and the microfluidic channel may have a height (e.g., H ch) at the proximal opening of about 30 microns to about 60 microns. The foregoing are examples only, and the width (e.g., W con or W con1) of the proximal opening (e.g., 234 or 274), the length (e.g., L con) of the connection region, and/or the width (e.g., W ch) of the microfluidic channel (e.g., 122 or 322), can be a value selected to be between any of the values listed above. Generally, however, the width (W con or W con1) of the proximal opening of the connection region of a sequestration pen is less than the width (W ch) of the microfluidic channel. In some embodiments, the width (W con or W con1) of the proximal opening is about 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 22%, 24%, 25%, or 30% of the width (W ch) of the microfluidic channel. That is, the width (W ch) of the microfluidic channel may be at least 2.5 times, 3.0 times, 3.5 times, 4.times, 4.5 times, 5.0 times, 6.0 times, 7.0 times, 8.0 times, 9.0 times or at least 10.0 times the width (W con or W con1) of the proximal opening of the connection region of the sequestration pen. In some embodiments, the size W C (e.g., cross-sectional width W ch, diameter, area, or the like) of the channel 122, 322, 618, 718 can be about one and a quarter (1.25), about one and a half (1.5), about two, about two and a half (2.5), about three (3), or more times the size W O (e.g., cross-sectional width W con, diameter, area, or the like) of a chamber opening, e.g., sequestration pen opening 234, 334, and the like. This can reduce the extent of secondary flow and the rate of diffusion (or diffusion flux) through the opening 234, 334 for materials diffusing from a selected chamber (e.g., like sequestration pens 224, 226 of FIG. 2B) into channel 122, 322, 618, 718 and subsequently re-entering a downstream or adjacent chamber (e.g., like sequestration pen 228). The rate of diffusion of a molecule (e.g., an analyte of interest, such as an antibody) is dependent on a number of factors, including (without limitation) temperature, viscosity of the medium, and the coefficient of diffusion D 0 of the molecule.

For example, the D 0 for an IgG antibody in aqueous solution at about 20°C is about 4.4x10-7 cm/sec, while the kinematic viscosity of cell culture medium is about 9x10-4 m/sec. Thus, an antibody in cell culture medium at about 20°C can have a rate of diffusion of about 0.5 microns/sec. Accordingly, in some embodiments, a time period for diffusion from a biological micro-object located within a sequestration pen such as 224, 226, 228, 324 into the channel 122, 322, 618, 718 can be about 10 minutes or less (e.g., about 9, 8, 7, 6, 5 minutes, or less). The time period for diffusion can be manipulated by changing parameters that influence the rate of diffusion. For example, the temperature of the media can be increased (e.g., to a physiological temperature such as about 37°C) or decreased (e.g., to about 15°C, 10°C, or 4°C) thereby increasing or decreasing the rate of diffusion, respectively. Alternatively, or in addition, the concentrations of solutes in the medium can be increased or decreased as discussed herein to isolate a selected pen from solutes from other upstream pens. Accordingly, in some variations, the width (e.g., W ch) of the microfluidic channel at the proximal opening to the connection region of a sequestration pen may be about 50 to 500 microns, about 50 to 300 microns, about 50 to 200 microns, about 70 to 500 microns, about to 70-300 microns, about 70 to 250 microns, about 70 to 200 microns, about 70 to 150 microns, about 70 to 100 microns, about 80 to 500 microns, about 80 to 300 microns, about 80 to 250 microns, about 80 to 200 microns, about 80 to 150 microns, about 90 to 500 microns, about 90 to 300 microns, about 90 to 250 microns, about 90 to 200 microns, about 90 to 150 microns, about 100 to 500 microns, about 100 to 300 microns, about 100 to 250 microns, about 100 to 200 microns, or about 100 to 150 microns. In some embodiments, the width W ch of the microfluidic channel at the proximal opening to the connection region of a sequestration pen may be about 70 to 250 microns, about 80 to 200 microns, or about 90 to 150 microns. The width W con of the opening of the chamber (e.g., sequestration pen) may be about to 100 microns; about 30 to 90 microns; or about 20 to 60 microns. In some embodiments, W ch is about 70-250 microns and W con is about 20 to 100 microns; W ch is about 80 to 200 microns and W con is about to 90 microns; W ch is about 90 to 150 microns, and W con is about 20 to 60 microns; or any combination of the widths of W ch and W con thereof. In some embodiments, the proximal opening (e.g., 234 or 334) of the connection region of a sequestration pen has a width (e.g., W con or W con1) that is 2.0 times or less (e.g., 2.0, 1.9, 1.8, 1.5, 1.3, 1.0, 0.8, 0.5, or 0.1 times) the height (e.g., H ch) of the flow region/ microfluidic channel at the proximal opening, or has a value that lies within a range defined by any two of the foregoing values. In some embodiments, the width W con1 of a proximal opening (e.g., 234 or 334) of a connection region of a sequestration pen may be the same as a width W con2 of the distal opening (e.g., 238 or 338) to the isolation region thereof. In some embodiments, the width W con1 of the proximal opening may be different than a width W con2 of the distal opening, and W con1 and/or W con2 may be selected from any of the values described for W con or W con1. In some embodiments, the walls (including a connection region wall) that define the proximal opening and distal opening may be substantially parallel with respect to each other. In some embodiments, the walls that define the proximal opening and distal opening may be selected to not be parallel with respect to each other. The length (e.g., L con) of the connection region can be about 1-600 microns, 5-550 microns, 10- 500 microns, 15-400 microns, 20-300 microns, 20-500 microns, 40-400 microns, 60-300 microns, 80-200 microns, about 100-150 microns, about 20-300 microns, about 20 -250 microns, about 20-2microns, about 20-150 microns, about 20-100 microns, about 30-250 microns, about 30-200 microns, about 30- 150 microns, about 30-100 microns, about 30-80 microns, about 30-50 microns, about 45-250 microns, about 45-200 microns, about 45-100 microns, about 45- 80 microns, about 45-60 microns, about 60-200 microns, about 60-150 microns, about 60-100 microns or about 60-80 microns. The foregoing are examples only, and length (e.g., L con) of a connection region can be selected to be a value that is between any of the values listed above. The connection region wall of a sequestration pen may have a length (e.g., L wall) that is at least 0.5 times, at least 0.6 times, at least 0.7 times, at least 0.8 times, at least 0.9 times, at least 1.0 times, at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.times, at least 2.0 times, at least 2.25 times, at least 2.5 times, at least 2.75 times, at least 3.0 times, or at least 3.5 times the width (e.g., W con or W con1) of the proximal opening of the connection region of the sequestration pen. In some embodiments, the connection region wall may have a length L wall of about 20-200 microns, about 20-150 microns, about 20-100 microns, about 20-80 microns, or about 20-50 microns. The foregoing are examples only, and a connection region wall may have a length L wall selected to be between any of the values listed above. A sequestration pen may have a length L s of about 40-600 microns, about 40-500 microns, about 40-400 microns, about 40-300 microns, about 40-200 microns, about 40-100 microns or about 40-80 microns. The foregoing are examples only, and a sequestration pen may have a length L s selected to be between any of the values listed above. According to some embodiments, a sequestration pen may have a specified height (e.g., H s). In some embodiments, a sequestration pen has a height H s of about 20 microns to about 200 microns (e.g., about 20 microns to about 150 microns, about 20 microns to about 100 microns, about 20 microns to about 60 microns, about 30 microns to about 150 microns, about 30 microns to about 100 microns, about 30 microns to about 60 microns, about 40 microns to about 150 microns, about 40 microns to about 100 microns, or about 40 microns to about 60 microns). The foregoing are examples only, and a sequestration pen can have a height H s selected to be between any of the values listed above.

The height H con of a connection region at a proximal opening of a sequestration pen can be a height within any of the following heights: 20-100 microns, 20-90 microns, 20-80 microns, 20-microns, 20-60 microns, 20-50 microns, 30-100 microns, 30-90 microns, 30-80 microns, 30-microns, 30-60 microns, 30-50 microns, 40-100 microns, 40-90 microns, 40-80 microns, 40-microns, 40-60 microns, or 40-50 microns. The foregoing are examples only, and the height H con of the connection region can be selected to be between any of the values listed above. Typically, the height H con of the connection region is selected to be the same as the height H ch of the microfluidic channel at the proximal opening of the connection region. Additionally, the height H s of the sequestration pen is typically selected to be the same as the height H con of a connection region and/or the height H ch of the microfluidic channel. In some embodiments, H s, H con, and H ch may be selected to be the same value of any of the values listed above for a selected microfluidic device. The isolation region can be configured to contain only one, two, three, four, five, or a similar relatively small number of micro-objects. In other embodiments, the isolation region may contain more than 10, more than 50 or more than 100 micro-objects. Accordingly, the volume of an isolation region can be, for example, at least 1x10, 1x10, 5x10, 8x10, 1x10, 2x10, 4x10, 6x10, 1x10, 3x10, 5x10 1x10, 5x10, or 8x10cubic microns, or more. The foregoing are examples only, and the isolation region can be configured to contain numbers of micro-objects and volumes selected to be between any of the values listed above (e.g., a volume between 1x10 cubic microns and 5x10 cubic microns, between 5x10 cubic microns and 1x10 cubic microns, between 1x10 cubic microns and 2x10 cubic microns, or between 2x10 cubic microns and 1x10cubic microns). According to some embodiments, a sequestration pen of a microfluidic device may have a specified volume. The specified volume of the sequestration pen (or the isolation region of the sequestration pen) may be selected such that a single cell or a small number of cells (e.g., 2-10 or 2-5) can rapidly condition the medium and thereby attain favorable (or optimal) growth conditions. In some embodiments, the sequestration pen has a volume of about 5x10, 6x10, 8x10, 1x10, 2x10, 4x10, 8x10, 1x10, 3x10, 5x10, or about 8x10 cubic microns, or more. In some embodiments, the sequestration pen has a volume of about 1 nanoliter to about 50 nanoliters, 2 nanoliters to about nanoliters, 2 nanoliters to about 20 nanoliters, about 2 nanoliters to about 15 nanoliters, or about nanoliters to about 10 nanoliters. The foregoing are examples only, and a sequestration pen can have a volume selected to be any value that is between any of the values listed above. According to some embodiments, the flow of fluidic medium within the microfluidic channel (e.g., 122 or 322) may have a specified maximum velocity (e.g., V max). In some embodiments, the maximum velocity (e.g., V max) may be set at around 0.2, 0.5, 0.7, 1.0, 1.3, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.7, 7.0, 7.5, 8.0, 8.5, 9.0, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, or 25 microliters/sec. The foregoing are examples only, and the flow of fluidic medium within the microfluidic channel can have a maximum velocity (e.g., V max) selected to be a value between any of the values listed above. The flow of fluidic medium within the microfluidic channel typically may be flowed at a rate less than the V max. While the V max may vary depending on the specific size and numbers of channel and sequestration pens opening thereto, a fluidic medium may be flowed at about 0.1 microliters/sec to about 20 microliters/sec; about 0.1 microliters/sec to about 15 microliters/sec; about 0.1 microliters/sec to about 12 microliters/sec, about 0.1 microliters/sec to about 10 microliters/sec; about 0.1 microliter/sec to about 7 microliters/sec without exceeding the V max. In some portions of a typical workflow, a flow rate of a fluidic medium may be about 0.1 microliters/sec; about 0.microliters/sec; about 1.0 microliters/sec; about 2.0 microliters/sec; about 3.0 microliters/sec; about 4.0 microliters/sec; about 5.0 microliters/sec; about 6.0 microliters/sec; about 7.0 microliters/sec; about 8.microliters/sec; about 9.0 microliters/sec; about 10.0 microliters/sec; about 11.0 microliters/sec; or any range defined by two of the foregoing values, e.g., 1-5 microliters/sec or 5-10 microliters/sec. The flow rate of a fluidic medium in the microfluidic channel may be equal to or less than about microliters/sec; about 10 microliters/sec; about 8 microliters/sec, or about 6 microliters/sec. In various embodiment, the microfluidic device has sequestration pens configured as in any of the embodiments discussed herein where the microfluidic device has about 5 to about 10 sequestration pens, about 10 to about 50 sequestration pens, about 25 to about 200 sequestration pens, about 100 to about 500 sequestration pens, about 200 to about 1000 sequestration pens, about 500 to about 15sequestration pens, about 1000 to about 2500 sequestration pens, about 2000 to about 5000 sequestration pens, about 3500 to about 7000 sequestration pens, about 5000 to about 10,0sequestration pens, about 7,500 to about 15,000 sequestration pens, about 12,500 to about 20,0sequestration pens, about 15,000 to about 25,000 sequestration pens, about 20,000 to about 30,0sequestration pens, about 25,000 to about 35,000 sequestration pens, about 30,000 to about 40,0sequestration pens, about 35,000 to about 45,000 sequestration pens, or about 40,000 to about 50,000 sequestration pens. The sequestration pens need not all be the same size and may include a variety of configurations (e.g., different widths, different features within the sequestration pen). Figure 2G illustrates a microfluidic device 280 according to one embodiment. The microfluidic device 280 illustrated in Figure 2G is a stylized diagram of a microfluidic device 100. In practice the microfluidic device 280 and its constituent circuit elements (e.g. channels 122 and chambers 128) would have the dimensions discussed herein. The microfluidic circuit 120 illustrated in Figure 2G has two ports 107, four distinct channels 122 and four distinct flow paths 106. The microfluidic device 280 further comprises a plurality of chambers opening off of each channel 122. In the microfluidic device illustrated in Figure 2G, the chambers have a geometry similar to the chambers illustrated in Figure 2C and thus, have both connection regions and isolation regions. Accordingly, the microfluidic circuit 120 includes both swept regions (e.g. channels 122 and portions of the connection regions 236 within the maximum penetration depth D p of the secondary flow 244) and non-swept regions (e.g. isolation regions 240 and portions of the connection regions 236 not within the maximum penetration depth D p of the secondary flow 244). Figures 3B through 3C shows various embodiments of system 150 which can be used to operate and observe microfluidic devices (e.g. 100, 200, 230, 250, 280, 290, 300, 400, 500, 900, 1000, 1100, 1200) according to the present disclosure. As illustrated in Figure 3B, the system 1can include a structure ("nest") 305 configured to hold a microfluidic device 320 ,or any other microfluidic device described herein. The nest 305 can include a socket 301 capable of interfacing with the microfluidic device 320 (e.g., an optically-actuated electrokinetic device 100) and providing electrical connections from power source 192 to microfluidic device 320. The nest 305 can further include an integrated electrical signal generation subsystem 304. The electrical signal generation subsystem 303 can be configured to supply a biasing voltage to socket 301 such that the biasing voltage is applied across a pair of electrodes in the microfluidic device 320 when it is being held by socket 301. Thus, the electrical signal generation subsystem 303 can be part of power source 192. The ability to apply a biasing voltage to microfluidic device 320 does not mean that a biasing voltage will be applied at all times when the microfluidic device 320 is held by the socket 301. Rather, in most cases, the biasing voltage will be applied intermittently, e.g., only as needed to facilitate the generation of electrokinetic forces, such as dielectrophoresis or electro-wetting, in the microfluidic device 320. As illustrated in Figure 3B, the nest 305 can include a printed circuit board assembly (PCBA) 322. The electrical signal generation subsystem 303 can be mounted on and electrically integrated into the PCBA 322. The exemplary support includes socket 301 mounted on PCBA 322, as well. Typically, the electrical signal generation subsystem 303 will include a waveform generator (not shown). The electrical signal generation subsystem 303 can further include an oscilloscope (not shown) and/or a waveform amplification circuit (not shown) configured to amplify a waveform received from the waveform generator. The oscilloscope, if present, can be configured to measure the waveform supplied to the microfluidic device 320 held by the socket 301. In certain embodiments, the oscilloscope measures the waveform at a location proximal to the microfluidic device 320 (and distal to the waveform generator), thus ensuring greater accuracy in measuring the waveform actually applied to the device. Data obtained from the oscilloscope measurement can be, for example, provided as feedback to the waveform generator, and the waveform generator can be configured to adjust its output based on such feedback. An example of a suitable combined waveform generator and oscilloscope is the Red Pitaya™. In certain embodiments, the nest 305 further comprises a controller 307, such as a microprocessor used to sense and/or control the electrical signal generation subsystem 303. Examples of suitable microprocessors include the Arduino™ microprocessors, such as the Arduino Nano™. The controller 307 may be used to perform functions and analysis or may communicate with an external master controller 154 (shown in Figure 1A) to perform functions and analysis. In the embodiment illustrated in Figure 3A the controller 307 communicates with a master controller 1through an interface 309 (e.g., a plug or connector). In some embodiments, the nest 305 can comprise an electrical signal generation subsystem 303 comprising a Red Pitaya™ waveform generator/oscilloscope unit ("Red Pitaya unit") and a waveform amplification circuit that amplifies the waveform generated by the Red Pitaya unit and passes the amplified voltage to the microfluidic device 320. In some embodiments, the Red Pitaya unit is configured to measure the amplified voltage at the microfluidic device 320 and then adjust its own output voltage as needed such that the measured voltage at the microfluidic device 320 is the desired value. In some embodiments, the waveform amplification circuit can have a +6.5V to -6.5V power supply generated by a pair of DC-DC converters mounted on the PCBA 322, resulting in a signal of up to 13 Vpp at the microfluidic device 320. As illustrated in Figure 3B, the support structure 305 (e.g., nest) can further include a thermal control subsystem 306. The thermal control subsystem 306 can be configured to regulate the temperature of microfluidic device 320 held by the support structure 305. For example, the thermal control subsystem 306 can include a Peltier thermoelectric device (not shown) and a cooling unit (not shown). The Peltier thermoelectric device can have a first surface configured to interface with at least one surface of the microfluidic device 320. The cooling unit can be, for example, a cooling block (not shown), such as a liquid-cooled aluminum block. A second surface of the Peltier thermoelectric device (e.g., a surface opposite the first surface) can be configured to interface with a surface of such a cooling block. The cooling block can be connected to a fluidic path 314 configured to circulate cooled fluid through the cooling block. In the embodiment illustrated in Figure 3A, the support structure 300 comprises an inlet 315 and an outlet 318 to receive cooled fluid from an external reservoir (not shown), introduce the cooled fluid into the fluidic path 314 and through the cooling block, and then return the cooled fluid to the external reservoir. In some embodiments, the Peltier thermoelectric device, the cooling unit, and/or the fluidic path 314 can be mounted on a casing 312 of the support structure 305. In some embodiments, the thermal control subsystem 306 is configured to regulate the temperature of the Peltier thermoelectric device so as to achieve a target temperature for the microfluidic device 320. Temperature regulation of the Peltier thermoelectric device can be achieved, for example, by a thermoelectric power supply, such as a Pololu™ thermoelectric power supply (Pololu Robotics and Electronics Corp.). The thermal control subsystem 306 can include a feedback circuit, such as a temperature value provided by an analog circuit. Alternatively, the feedback circuit can be provided by a digital circuit. In some embodiments, the nest 305 can include a thermal control subsystem 306 with a feedback circuit that is an analog voltage divider circuit (not shown) which includes a resistor (e.g., with resistance 1 kOhm+/-0.1 %, temperature coefficient +/-0.02 ppm/C0) and a NTC thermistor (e.g., with nominal resistance 1 kOhm+/-0.01 %). In some instances, the thermal control subsystem 306 measures the voltage from the feedback circuit and then uses the calculated temperature value as input to an on-board PID control loop algorithm. Output from the PID control loop algorithm can drive, for example, both a directional and a pulse-width-modulated signal pin on a Pololu™ motor drive (not shown) to actuate the thermoelectric power supply, thereby controlling the Peltier thermoelectric device. The nest 305 can include a serial port 324 which allows the microprocessor of the controller 308 to communicate with an external master controller 154 via the interface 310 (not shown). In addition, the microprocessor of the controller 307 can communicate (e.g., via a Plink tool (not shown)) with the electrical signal generation subsystem 303 and thermal control subsystem 306. Thus, via the combination of the controller 307, the interface 309, and the serial port 324, the electrical signal generation subsystem 303 and the thermal control subsystem 306 can communicate with the external master controller 154. In this manner, the master controller 154 can, among other things, assist the electrical signal generation subsystem 304 by performing scaling calculations for output voltage adjustments. A Graphical User Interface (GUI) (not shown) provided via a display device 170 coupled to the external master controller 154, can be configured to plot temperature and waveform data obtained from the thermal control subsystem 306 and the electrical signal generation subsystem 303, respectively. Alternatively, or in addition, the GUI can allow for updates to the controller 307, the thermal control subsystem 306, and the electrical signal generation subsystem 303. As discussed above, system 150 can include an imaging device 194. In some embodiments, the imaging device 194 comprises a light modulating subsystem 350 (See Figure 3C). The light modulating subsystem 335 can include a digital mirror device (DMD) or a microshutter array system (MSA), either of which can be configured to receive light from a light source 332 and transmits a subset of the received light into an optical train of microscope 350. Alternatively, the light modulating subsystem 335 can include a device that produces its own light (and thus dispenses with the need for a light source 332), such as an organic light emitting diode display (OLED), a liquid crystal on silicon (LCOS) device, a ferroelectric liquid crystal on silicon device (FLCOS), or a transmissive liquid crystal display (LCD). The light modulating subsystem 335 can be, for example, a projector. Thus, the light modulating subsystem 350 can be capable of emitting both structured and unstructured light. In certain embodiments, imaging module 164 and/or motive module 162 of system 150 can control the light modulating subsystem 330. In certain embodiments, the imaging device 194 further comprises a microscope 350. In such embodiments, the nest 305 and light modulating subsystem 335 can be individually configured to be mounted on the microscope 350. The microscope 350 can be, for example, a standard research-grade light microscope or fluorescence microscope. Thus, the nest 305 can be configured to be mounted on the stage 344 of the microscope 350 and/or the light modulating subsystem 335 can be configured to mount on a port of microscope 350. In other embodiments, the nest 305 and the light modulating subsystem 335 described herein can be integral components of microscope 350. In certain embodiments, the microscope 350 can further include one or more detectors 348. In some embodiments, the detector 348 is controlled by the imaging module 164. The detector 3can include an eye piece, a charge-coupled device (CCD), a camera (e.g., a digital camera), or any combination thereof. If at least two detectors 348 are present, one detector can be, for example, a fast-frame-rate camera while the other detector can be a high sensitivity camera. Furthermore, the microscope 350 can include an optical train configured to receive reflected and/or emitted light from the microfluidic device 320 and focus at least a portion of the reflected and/or emitted light on the one or more detectors 348. The optical train of the microscope can also include different tube lenses (not shown) for the different detectors, such that the final magnification on each detector can be different. In certain embodiments, imaging device 194 is configured to use at least two light sources. For example, a first light source 332 can be used to produce structured light (e.g., via the light modulating subsystem 335) and a second light source 334 can be used to provide unstructured light. The first light source 332 can produce structured light for optically-actuated electrokinesis and/or fluorescent excitation, and the second light source 333 can be used to provide bright field illumination. In these embodiments, the motive module 164 can be used to control the first light source 332 and the imaging module 164 can be used to control the second light source 333. The optical train of the microscope 350 can be configured to (1) receive structured light from the light modulating subsystem 335 and focus the structured light on at least a first region in a microfluidic device, such as an optically-actuated electrokinetic device, when the device is being held by the nest 305, and (2) receive reflected and/or emitted light from the microfluidic device and focus at least a portion of such reflected and/or emitted light onto detector 348. The optical train can be further configured to receive unstructured light from a second light source and focus the unstructured light on at least a second region of the microfluidic device, when the device is held by the nest 305. In certain embodiments, the first and second regions of the microfluidic device can be overlapping regions. For example, the first region can be a subset of the second region. In other embodiments, the second light source 333 may additionally or alternatively include a laser, which may have any suitable wavelength of light. The representation of the optical system shown in Figure 3C is a schematic representation only, and the optical system may include additional filters, notch filters, lenses and the like. When the second light source 333 includes one or more light source(s) for brightfield and/or fluorescent excitation, as well as laser illumination the physical arrangement of the light source(s) may vary from that shown in Figure 3C, and the laser illumination may be introduced at any suitable physical location within the optical system. The schematic locations of light source 333 and light source 332/light modulating subsystem 335 may be interchanged as well. In Figure 3C, the first light source 332 is shown supplying light to a light modulating subsystem 335, which provides structured light to the optical train of the microscope 350 of system 355 (not shown). The second light source 333 is shown providing unstructured light to the optical train via a beam splitter 335. Structured light from the light modulating subsystem 335 and unstructured light from the second light source 333 travel from the beam splitter 337 through the optical train together to reach a second beam splitter (or dichroic filter 339, depending on the light provided by the light modulating subsystem 335), where the light gets reflected down through the objective 340 to the sample plane 342. Reflected and/or emitted light from the sample plane 342 then travels back up through the objective 340, through the beam splitter and/or dichroic filter 339, and to a dichroic filter 346. Only a fraction of the light reaching dichroic filter 346 passes through and reaches the detector 348. In some embodiments, the second light source 333 emits blue light. With an appropriate dichroic filter 346, blue light reflected from the sample plane 342 is able to pass through dichroic filter 346 and reach the detector 348. In contrast, structured light coming from the light modulating subsystem 335 gets reflected from the sample plane 342, but does not pass through the dichroic filter 346. In this example, the dichroic filter 346 is filtering out visible light having a wavelength longer than 495 nm. Such filtering out of the light from the light modulating subsystem 335 would only be complete (as shown) if the light emitted from the light modulating subsystem did not include any wavelengths shorter than 495 nm. In practice, if the light coming from the light modulating subsystem 335 includes wavelengths shorter than 495 nm (e.g., blue wavelengths), then some of the light from the light modulating subsystem would pass through filter 346 to reach the detector 348. In such an embodiment, the filter 346 acts to change the balance between the amount of light that reaches the detector 348 from the first light source 332 and the second light source 333. This can be beneficial if the first light source 332 is significantly stronger than the second light source 333. In other embodiments, the second light source 333 can emit red light, and the dichroic filter 346 can filter out visible light other than red light (e.g., visible light having a wavelength shorter than 6nm). Coating solutions and coating agents. Without intending to be limited by theory, maintenance of a biological micro-object (e.g., a biological cell) within a microfluidic device (e.g., a DEP-configured and/or EW-configured microfluidic device) may be facilitated (i.e., the biological micro-object exhibits increased viability, greater expansion and/or greater portability within the microfluidic device) when at least one or more inner surfaces of the microfluidic device have been conditioned or coated so as to present a layer of organic and/or hydrophilic molecules that provides the primary interface between the microfluidic device and biological micro-object(s) maintained therein. In some embodiments, one or more of the inner surfaces of the microfluidic device (e.g. the inner surface of the electrode activation substrate of a DEP-configured microfluidic device, the cover of the microfluidic device, and/or the surfaces of the circuit material) may be treated with or modified by a coating solution and/or coating agent to generate the desired layer of organic and/or hydrophilic molecules. The coating may be applied before or after introduction of biological micro-object(s), or may be introduced concurrently with the biological micro-object(s). In some embodiments, the biological micro-object(s) may be imported into the microfluidic device in a fluidic medium that includes one or more coating agents. In other embodiments, the inner surface(s) of the microfluidic device (e.g., a DEP-configured microfluidic device) are treated or "primed" with a coating solution comprising a coating agent prior to introduction of the biological micro-object(s) into the microfluidic device. In some embodiments, at least one surface of the microfluidic device includes a coating material that provides a layer of organic and/or hydrophilic molecules suitable for maintenance and/or expansion of biological micro-object(s) (e.g. provides a conditioned surface as described below). In some embodiments, substantially all the inner surfaces of the microfluidic device include the coating material. The coated inner surface(s) may include the surface of a flow region (e.g., channel), chamber, or chamber, or a combination thereof. In some embodiments, each of a plurality of chambers has at least one inner surface coated with coating materials. In other embodiments, each of a plurality of flow regions or channels has at least one inner surface coated with coating materials. In some embodiments, at least one inner surface of each of a plurality of chambers and each of a plurality of channels is coated with coating materials.

Coating agent/Solution. Any convenient coating agent/coating solution can be used, including but not limited to: serum or serum factors, bovine serum albumin (BSA), polymers, detergents, enzymes, and any combination thereof. Polymer-based coating materials . The at least one inner surface may include a coating material that comprises a polymer. The polymer may be covalently or non-covalently bound (or may be non-specifically adhered) to the at least one surface. The polymer may have a variety of structural motifs, such as found in block polymers (and copolymers), star polymers (star copolymers), and graft or comb polymers (graft copolymers), all of which may be suitable for the methods disclosed herein. The polymer may include a polymer including alkylene ether moieties. A wide variety of alkylene ether containing polymers may be suitable for use in the microfluidic devices described herein. One non-limiting exemplary class of alkylene ether containing polymers are amphiphilic nonionic block copolymers which include blocks of polyethylene oxide (PEO) and polypropylene oxide (PPO) subunits in differing ratios and locations within the polymer chain. Pluronic® polymers (BASF) are block copolymers of this type and are known in the art to be suitable for use when in contact with living cells. The polymers may range in average molecular mass M w from about 2000Da to about 20KDa. In some embodiments, the PEO-PPO block copolymer can have a hydrophilic-lipophilic balance (HLB) greater than about 10 (e.g. 12-18). Specific Pluronic® polymers useful for yielding a coated surface include Pluronic® L44, L64, P85, and F127 (including F127NF). Another class of alkylene ether containing polymers is polyethylene glycol (PEG M w <100,000Da) or alternatively polyethylene oxide (PEO, M w>100,000). In some embodiments, a PEG may have an M w of about 1000Da, 5000Da, 10,000Da or 20,000Da. In other embodiments, the coating material may include a polymer containing carboxylic acid moieties. The carboxylic acid subunit may be an alkyl, alkenyl or aromatic moiety containing subunit. One non-limiting example is polylactic acid (PLA). In other embodiments, the coating material may include a polymer containing phosphate moieties, either at a terminus of the polymer backbone or pendant from the backbone of the polymer. In yet other embodiments, the coating material may include a polymer containing sulfonic acid moieties. The sulfonic acid subunit may be an alkyl, alkenyl or aromatic moiety containing subunit. One non-limiting example is polystyrene sulfonic acid (PSSA) or polyanethole sulfonic acid. In further embodiments, the coating material may include a polymer including amine moieties. The polyamino polymer may include a natural polyamine polymer or a synthetic polyamine polymer. Examples of natural polyamines include spermine, spermidine, and putrescine. In other embodiments, the coating material may include a polymer containing saccharide moieties. In a non-limiting example, polysaccharides such as xanthan gum or dextran may be suitable to form a material which may reduce or prevent cell sticking in the microfluidic device. For example, a dextran polymer having a size about 3kDa may be used to provide a coating material for a surface within a microfluidic device. In other embodiments, the coating material may include a polymer containing nucleotide moieties, i.e. a nucleic acid, which may have ribonucleotide moieties or deoxyribonucleotide moieties, providing a polyelectrolyte surface. The nucleic acid may contain only natural nucleotide moieties or may contain unnatural nucleotide moieties which comprise nucleobase, ribose or phosphate moiety analogs such as 7-deazaadenine, pentose, methyl phosphonate or phosphorothioate moieties without limitation. In yet other embodiments, the coating material may include a polymer containing amino acid moieties. The polymer containing amino acid moieties may include a natural amino acid containing polymer or an unnatural amino acid containing polymer, either of which may include a peptide, a polypeptide or a protein. In one non-limiting example, the protein may be bovine serum albumin (BSA) and/or serum (or a combination of multiple different sera) comprising albumin and/or one or more other similar proteins as coating agents. The serum can be from any convenient source, including but not limited to fetal calf serum, sheep serum, goat serum, horse serum, and the like. In certain embodiments, BSA in a coating solution is present in a concentration from about 1 mg/mL to about 100 mg/mL, including 5 mg/mL, 10 mg/mL, 20 mg/mL, 30 mg/mL, 40 mg/mL, 50 mg/mL, mg/mL, 70 mg/mL, 80 mg/mL, 90 mg/mL, or more or anywhere in between. In certain embodiments, serum in a coating solution may be present in a concentration of about 20% (v/v) to about 50% v/v, including 25%, 30%, 35%, 40%, 45%, or more or anywhere in between. In some embodiments, BSA may be present as a coating agent in a coating solution at 5 mg/mL, whereas in other embodiments, BSA may be present as a coating agent in a coating solution at 70 mg/mL. In certain embodiments, serum is present as a coating agent in a coating solution at 30%. In some embodiments, an extracellular matrix (ECM) protein may be provided within the coating material for optimized cell adhesion to foster cell growth. A cell matrix protein, which may be included in a coating material, can include, but is not limited to, a collagen, an elastin, an RGD-containing peptide (e.g. a fibronectin), or a laminin. In yet other embodiments, growth factors, cytokines, hormones or other cell signaling species may be provided within the coating material of the microfluidic device. In some embodiments, the coating material may include a polymer containing more than one of alkylene oxide moieties, carboxylic acid moieties, sulfonic acid moieties, phosphate moieties, saccharide moieties, nucleotide moieties, or amino acid moieties. In other embodiments, the polymer conditioned surface may include a mixture of more than one polymer each having alkylene oxide moieties, carboxylic acid moieties, sulfonic acid moieties, phosphate moieties, saccharide moieties, nucleotide moieties, and/or amino acid moieties, which may be independently or simultaneously incorporated into the coating material. Covalently linked coating materials . In some embodiments, the at least one inner surface includes covalently linked molecules that provide a layer of organic and/or hydrophilic molecules suitable for maintenance/expansion of biological micro-object(s) within the microfluidic device, providing a conditioned surface for such cells. The covalently linked molecules include a linking group, wherein the linking group is covalently linked to one or more surfaces of the microfluidic device, as described below. The linking group is also covalently linked to a moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for maintenance/expansion of biological micro-object(s). In some embodiments, the covalently linked moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for maintenance/expansion of biological micro-object(s) may include alkyl or fluoroalkyl (which includes perfluoroalkyl) moieties; mono- or polysaccharides (which may include but is not limited to dextran); alcohols (including but not limited to propargyl alcohol); polyalcohols, including but not limited to polyvinyl alcohol; alkylene ethers, including but not limited to polyethylene glycol; polyelectrolytes ( including but not limited to polyacrylic acid or polyvinyl phosphonic acid); amino groups (including derivatives thereof, such as, but not limited to alkylated amines, hydroxyalkylated amino group, guanidinium, and heterocylic groups containing an unaromatized nitrogen ring atom, such as, but not limited to morpholinyl or piperazinyl); carboxylic acids including but not limited to propiolic acid (which may provide a carboxylate anionic surface); phosphonic acids, including but not limited to ethynyl phosphonic acid (which may provide a phosphonate anionic surface); sulfonate anions; carboxybetaines; sulfobetaines; sulfamic acids; or amino acids. In various embodiments, the covalently linked moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for maintenance/expansion of biological micro- object(s) in the microfluidic device may include non-polymeric moieties such as an alkyl moiety, a substituted alkyl moiety, such as a fluoroalkyl moiety (including but not limited to a perfluoroalkyl moiety), amino acid moiety, alcohol moiety, amino moiety, carboxylic acid moiety, phosphonic acid moiety, sulfonic acid moiety, sulfamic acid moiety, or saccharide moiety. Alternatively, the covalently linked moiety may include polymeric moieties, which may be any of the moieties described above. In some embodiments, the covalently linked alkyl moiety may comprise carbon atoms forming a linear chain (e.g., a linear chain of at least 10 carbons, or at least 14, 16, 18, 20, 22, or more carbons) and may be an unbranched alkyl moiety. In some embodiments, the alkyl group may include a substituted alkyl group (e.g., some of the carbons in the alkyl group can be fluorinated or perfluorinated). In some embodiments, the alkyl group may include a first segment, which may include a perfluoroalkyl group, joined to a second segment, which may include a non-substituted alkyl group, where the first and second segments may be joined directly or indirectly (e.g., by means of an ether linkage). The first segment of the alkyl group may be located distal to the linking group, and the second segment of the alkyl group may be located proximal to the linking group. In other embodiments, the covalently linked moiety may include at least one amino acid, which may include more than one type of amino acid. Thus, the covalently linked moiety may include a peptide or a protein. In some embodiments, the covalently linked moiety may include an amino acid which may provide a zwitterionic surface to support cell growth, viability, portability, or any combination thereof. In other embodiments, the covalently linked moiety may include at least one alkylene oxide moiety, and may include any alkylene oxide polymer as described above. One useful class of alkylene ether containing polymers is polyethylene glycol (PEG M w <100,000Da) or alternatively polyethylene oxide (PEO, M w>100,000). In some embodiments, a PEG may have an M w of about 1000Da, 5000Da, 10,000Da or 20,000Da. The covalently linked moiety may include one or more saccharides. The covalently linked saccharides may be mono-, di-, or polysaccharides. The covalently linked saccharides may be modified to introduce a reactive pairing moiety which permits coupling or elaboration for attachment to the surface. Exemplary reactive pairing moieties may include aldehyde, alkyne or halo moieties. A polysaccharide may be modified in a random fashion, wherein each of the saccharide monomers may be modified or only a portion of the saccharide monomers within the polysaccharide are modified to provide a reactive pairing moiety that may be coupled directly or indirectly to a surface. One exemplar may include a dextran polysaccharide, which may be coupled indirectly to a surface via an unbranched linker. The covalently linked moiety may include one or more amino groups. The amino group may be a substituted amine moiety, guanidine moiety, nitrogen-containing heterocyclic moiety or heteroaryl moiety. The amino containing moieties may have structures permitting pH modification of the environment within the microfluidic device, and optionally, within the chambers and/or flow regions (e.g., channels). The coating material providing a conditioned surface may comprise only one kind of covalently linked moiety or may include more than one different kind of covalently linked moiety. For example, the fluoroalkyl conditioned surfaces (including perfluoroalkyl) may have a plurality of covalently linked moieties which are all the same, e.g., having the same linking group and covalent attachment to the surface, the same overall length, and the same number of fluoromethylene units comprising the fluoroalkyl moiety. Alternatively, the coating material may have more than one kind of covalently linked moiety attached to the surface. For example, the coating material may include molecules having covalently linked alkyl or fluoroalkyl moieties having a specified number of methylene or fluoromethylene units and may further include a further set of molecules having charged moieties covalently attached to an alkyl or fluoroalkyl chain having a greater number of methylene or fluoromethylene units, which may provide capacity to present bulkier moieties at the coated surface. In this instance, the first set of molecules having different, less sterically demanding termini and fewer backbone atoms can help to functionalize the entire substrate surface and thereby prevent undesired adhesion or contact with the silicon/silicon oxide, hafnium oxide or alumina making up the substrate itself. In another example, the covalently linked moieties may provide a zwitterionic surface presenting alternating charges in a random fashion on the surface. Conditioned surface properties . Aside from the composition of the conditioned surface, other factors such as physical thickness of the hydrophobic material can impact DEP force. Various factors can alter the physical thickness of the conditioned surface, such as the manner in which the conditioned surface is formed on the substrate (e.g. vapor deposition, liquid phase deposition, spin coating, flooding, and electrostatic coating). In some embodiments, the conditioned surface has a thickness of about 1nm to about 10nm; about 1 nm to about 7 nm; about 1nm to about 5nm; or any individual value therebetween. In other embodiments, the conditioned surface formed by the covalently linked moieties may have a thickness of about 10 nm to about 50 nm. In various embodiments, the conditioned surface prepared as described herein has a thickness of less than 10nm. In some embodiments, the covalently linked moieties of the conditioned surface may form a monolayer when covalently linked to the surface of the microfluidic device (e.g., a DEP configured substrate surface) and may have a thickness of less than 10 nm (e.g., less than 5 nm, or about 1.5 to 3.0 nm). These values are in contrast to that of a surface prepared by spin coating, for example, which may typically have a thickness of about 30nm. In some embodiments, the conditioned surface does not require a perfectly formed monolayer to be suitably functional for operation within a DEP-configured microfluidic device. In various embodiments, the coating material providing a conditioned surface of the microfluidic device may provide desirable electrical properties. Without intending to be limited by theory, one factor that impacts robustness of a surface coated with a particular coating material is intrinsic charge trapping. Different coating materials may trap electrons, which can lead to breakdown of the coating material. Defects in the coating material may increase charge trapping and lead to further breakdown of the coating material. Similarly, different coating materials have different dielectric strengths (i.e. the minimum applied electric field that results in dielectric breakdown), which may impact charge trapping. In certain embodiments, the coating material can have an overall structure (e.g., a densely-packed monolayer structure) that reduces or limits that amount of charge trapping. In addition to its electrical properties, the conditioned surface may also have properties that are beneficial in use with biological molecules. For example, a conditioned surface that contains fluorinated (or perfluorinated) carbon chains may provide a benefit relative to alkyl-terminated chains in reducing the amount of surface fouling. Surface fouling, as used herein, refers to the amount of indiscriminate material deposition on the surface of the microfluidic device, which may include permanent or semi-permanent deposition of biomaterials such as protein and its degradation products, nucleic acids and respective degradation products and the like. Unitary or Multi-part conditioned surface . The covalently linked coating material may be formed by reaction of a molecule which already contains the moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for maintenance/expansion of biological micro-object(s) in the microfluidic device, as is described below. Alternatively, the covalently linked coating material may be formed in a two-part sequence by coupling the moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for maintenance/expansion of biological micro-object(s) to a surface modifying ligand that itself has been covalently linked to the surface. Methods of preparing a covalently linked coating material . In some embodiments, a coating material that is covalently linked to the surface of a microfluidic device (e.g., including at least one surface of the chambers and/or flow regions) has a structure of Formula 1 or Formula 2. When the coating material is introduced to the surface in one step, it has a structure of Formula 1, while when the coating material is introduced in a multiple step process, it has a structure of Formula 2. or Formula 1 Formula The coating material may be linked covalently to oxides of the surface of a DEP-configured or EW- configured substrate. The DEP- or EW- configured substrate may comprise silicon, silicon oxide, alumina, or hafnium oxide. Oxides may be present as part of the native chemical structure of the substrate or may be introduced as discussed below. The coating material may be attached to the oxides via a linking group ("LG"), which may be a siloxy or phosphonate ester group formed from the reaction of a siloxane or phosphonic acid group with the oxides. The moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for maintenance/expansion of biological micro-object(s) in the microfluidic device can be any of the moieties described herein. The linking group LG may be directly or indirectly connected to the moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for maintenance/expansion of biological micro-object(s) in the microfluidic device. When the linking group LG is directly connected to the moiety, optional linker ("L") is not present and n is 0. When the linking group LG is indirectly connected to the moiety, linker L is present and n is 1. The linker L may have a linear portion where a backbone of the linear portion may include 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur and/or phosphorus atoms, subject to chemical bonding limitations as is known in the art. It may be interrupted with any combination of one or more moieties, which may be chosen from ether, amino, carbonyl, amido, and/or phosphonate groups, arylene, heteroarylene, or heterocyclic groups. In some embodiments, the backbone of the linker L may include 10 to 20 atoms. In other embodiments, the backbone of the linker L may include about 5 atoms to about 200 atoms; about 10 atoms to about 80 atoms; about 10 atoms to about 50 atoms; or about 10 atoms to about 40 atoms. In some embodiments, the backbone atoms are all carbon atoms. In some embodiments, the moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for maintenance/expansion of biological micro-object(s) may be added to the surface of the substrate in a multi-step process, and has a structure of Formula 2, as shown above. The moiety may be any of the moieties described above. In some embodiments, the coupling group CG represents the resultant group from reaction of a reactive moiety R x and a reactive pairing moiety R px (i.e., a moiety configured to react with the reactive moiety R x). For example, one typical coupling group CG may include a carboxamidyl group, which is the result of the reaction of an amino group with a derivative of a carboxylic acid, such as an activated ester, an acid chloride or the like. Other CG may include a triazolylene group, a carboxamidyl, thioamidyl, an oxime, a mercaptyl, a disulfide, an ether, or alkenyl group, or any other suitable group that may be formed upon reaction of a reactive moiety with its respective reactive pairing moiety. The coupling group CG may be located at the second end (i.e., the end proximal to the moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for maintenance/expansion of biological micro-object(s) in the microfluidic device) of linker L, which may include any combination of elements as described above. In some other embodiments, the coupling group CG may interrupt the backbone of the linker L. When the coupling group CG is triazolylene, it may be the product resulting from a Click coupling reaction and may be further substituted (e.g., a dibenzocylcooctenyl fused triazolylene group). In some embodiments, the coating material (or surface modifying ligand) is deposited on the inner surfaces of the microfluidic device using chemical vapor deposition. The vapor deposition process can be optionally improved, for example, by pre-cleaning the cover 110, the microfluidic circuit material 116, and/or the substrate (e.g., the inner surface 208 of the electrode activation substrate 206 of a DEP-configured substrate, or a dielectric layer of the support structure 104 of an EW-configured substrate), by exposure to a solvent bath, sonication or a combination thereof. Alternatively, or in addition, such pre-cleaning can include treating the cover 110, the microfluidic circuit material 116, and/or the substrate in an oxygen plasma cleaner, which can remove various impurities, while at the same time introducing an oxidized surface (e.g. oxides at the surface, which may be covalently modified as described herein). Alternatively, liquid-phase treatments, such as a mixture of hydrochloric acid and hydrogen peroxide or a mixture of sulfuric acid and hydrogen peroxide (e.g., piranha solution, which may have a ratio of sulfuric acid to hydrogen peroxide from about 3:1 to about 7:1) may be used in place of an oxygen plasma cleaner. In some embodiments, vapor deposition is used to coat the inner surfaces of the microfluidic device 200 after the microfluidic device 200 has been assembled to form an enclosure 102 defining a microfluidic circuit 120. Without intending to be limited by theory, depositing such a coating material on a fully-assembled microfluidic circuit 120 may be beneficial in preventing delamination caused by a weakened bond between the microfluidic circuit material 116 and the electrode activation substrate 206 dielectric layer and/or the cover 110. In embodiments where a two-step process is employed the surface modifying ligand may be introduced via vapor deposition as described above, with subsequent introduction of the moiety configured provide a layer of organic and/or hydrophilic molecules suitable for maintenance/expansion of biological micro-object(s). The subsequent reaction may be performed by exposing the surface modified microfluidic device to a suitable coupling reagent in solution. Figure 2H depicts a cross-sectional view of a microfluidic device 290 having an exemplary covalently linked coating material providing a conditioned surface. As illustrated, the coating materials 298 (shown schematically) can comprise a monolayer of densely-packed molecules covalently bound to both the inner surface 294 of a base 286, which may be a DEP substrate, and the inner surface 292 of a cover 288 of the microfluidic device 290. The coating material 298 can be disposed on substantially all inner surfaces 294, 292 proximal to, and facing inwards towards, the enclosure 284 of the microfluidic device 290, including, in some embodiments and as discussed above, the surfaces of microfluidic circuit material (not shown) used to define circuit elements and/or structures within the microfluidic device 290. In alternate embodiments, the coating material 298 can be disposed on only one or some of the inner surfaces of the microfluidic device 290. In the embodiment shown in Figure 2H, the coating material 298 can include a monolayer of organosiloxane molecules, each molecule covalently bonded to the inner surfaces 292, 294 of the microfluidic device 290 via a siloxy linker 296. Any of the above-discussed coating materials 2can be used (e.g. an alkyl-terminated, a fluoroalkyl terminated moiety, a PEG- terminated moiety, a dextran terminated moiety, or a terminal moiety containing positive or negative charges for the organosiloxy moieties), where the terminal moiety is disposed at its enclosure-facing terminus (i.e. the portion of the monolayer of the coating material 298 that is not bound to the inner surfaces 292, 294 and is proximal to the enclosure 284). In other embodiments, the coating material 298 used to coat the inner surface(s) 292, 294 of the microfluidic device 290 can include anionic, cationic, or zwitterionic moieties, or any combination thereof. Without intending to be limited by theory, by presenting cationic moieties, anionic moieties, and/or zwitterionic moieties at the inner surfaces of the enclosure 284 of the microfluidic circuit 120, the coating material 298 can form strong hydrogen bonds with water molecules such that the resulting water of hydration acts as a layer (or "shield") that separates the biological micro-objects from interactions with non-biological molecules (e.g., the silicon and/or silicon oxide of the substrate). In addition, in embodiments in which the coating material 298 is used in conjunction with coating agents, the anions, cations, and/or zwitterions of the coating material 2can form ionic bonds with the charged portions of non-covalent coating agents (e.g. proteins in solution) that are present in a medium 180 (e.g. a coating solution) in the enclosure 284. In still other embodiments, the coating material may comprise or be chemically modified to present a hydrophilic coating agent at its enclosure-facing terminus. In some embodiments, the coating material may include an alkylene ether containing polymer, such as PEG. In some embodiments, the coating material may include a polysaccharide, such as dextran. Like the charged moieties discussed above (e.g., anionic, cationic, and zwitterionic moieties), the hydrophilic coating agent can form strong hydrogen bonds with water molecules such that the resulting water of hydration acts as a layer (or "shield") that separates the biological micro-objects from interactions with non-biological molecules (e.g., the silicon and/or silicon oxide of the substrate).

Further details of appropriate coating treatments and modifications may be found at U.S. Application Serial No. 15/135,707, filed on April 22, 2016, and is incorporated by reference in its entirety. Additional system components for maintenance of viability of cells within the chambers of the microfluidic device . In order to promote growth and/or expansion of cell populations, environmental conditions conducive to maintaining functional cells may be provided by additional components of the system. For example, such additional components can provide nutrients, cell growth signaling species, pH modulation, gas exchange, temperature control, and removal of waste products from cells. Assaying an analyte secreted by a biological micro-object. In some embodiments, the disclosure provides methods, systems and devices for quantifying a biological molecule present in chambers. In some embodiments, the biological molecule is a secreted analyte of a biological cell or any other biological micro-organism capable of producing a secreted analyte. In the bioproduction industry, one severe problem is the expense, time and difficulty in identifying clonal populations having desired levels of production and growth habits when employing the currently available instrumentation and workflows. For example, developing a new antibody production line can take many months of work and cost millions of dollars in personnel, equipment and materials. The ability to screen and identify promising clones within a microfluidic device, very early in expanding populations, such as 3, 4, 5, 6, or 7 days after seeding individual founding cells, as described herein, can offer significant time and cost advantages. It has been discovered by Applicant, that the nanofluidic environment, particularly one based on chambers, as described herein, provides exemplary isolation of clonal populations from each other, permitting the ability to obtain assay results from each individual clonal population without contamination from other clonal populations located within the microfluidic device. It has also been discovered that assays to determine the relative or absolute amount of a secreted analyte using the methods described herein, even when performed at an early stage of clonal expansion, can be correlated to production of the desired secreted analyte at more typical macroscale scale of expansion (e.g, shake flasks, etc.). Further, the ability to screen individual clones at such an early stage can also permit identification of desired clones meeting specific requirements of growth rate and/or more robust production (for example, highly productive clones which are more resistant to levels of a material in the culturing environment such as metabolic waste products or exhausted nutrients). Another advantage discovered by Applicant is that more complete exploration of a plurality of cells as potential founding cells for a clonal population can be made without use of excessive resources because the nanofluidic chambers (e.g., chambers) described here permit simultaneous growth/assay for up to thousands of individual founding cells at the same time in extremely small volumes. Additionally, the nanofluidic environment described here permits examination of the effects of specific conditions upon cells, with feedback from repeated assays. For example, conditions and materials, such as culture medium, more closely related to large scale production of a secreted product of a cell (an analyte in the methods herein) may be used to find and characterize the most suitable clones for further examination. In another example, diverse stimulation protocols for B-cell antibody stimulation may be examined in a more reproducible manner, and may be assayed in order to more comparably assess the benefits of one protocol over another. Detection and quantification using diffusion profiles. As described herein, the amount of a secreted analyte of a biological micro-object may be quantified using a reporter molecule that binds to the secreted analyte. The reporter molecule includes a binding component that binds the secreted analyte to be quantified and a signal component that is used to detect a quantity of the reporter molecule. The reporter molecule has a higher diffusion rate in its unbound state (e.g., not bound to a secreted analyte) than in its bound state (e.g., bound to one or more molecules of the secreted analyte). In some embodiments, the difference in the diffusion rate between the unbound and bound reporter molecules will be a function of the size of secreted analyte molecule(s) that the reporter molecule binds to. In some embodiments, the reporter molecule may bind the secreted analyte in a conformation that slows the rate of diffusion. For example, the reporter molecule may bind multiple copies of the secreted analyte in a conformation in which the secreted analyte is aggregated and diffuses slowly due, in part, to its conformation. The methods described herein exploit the differences in the rate of diffusion between the reporter molecule (unbound) and the bound reporter molecule:analyte complex (RMSA) to quantify the amount of the secreted analyte. Diffusion assay under flow conditions in the microfluidic channel. Figures 4A-4C illustrate an assay according to some embodiments of the disclosure. In Figure 4A, reporter molecules 412, each having a detectable label, are introduced into the microfluidic channel 122 by flowing a fluid containing a concentration of the reporter molecules 412 within flow 242 into the channel 122 of microfluidic device 400. Chambers 424, 426, 428 are each fluidically connected to the microfluidic channel 122 containing various numbers of cells 402, 404, 406 secreting a biological analyte 410. Each of chambers 424, 426, 428 include a connection region 436 and an isolation region 440 (chamber 424 is the only pen so labeled, just for clarity). The connection region 436 and isolation region 440 have properties as described above, and limit the contact of materials introduced into the channel 122 (e.g., within isolation region 440, materials flowing within the channel 122 may enter the isolation region only by diffusion, not by flow directly into the isolation region.) At the time point illustrated in Figure 4A, the molecules of the secreted analyte 410 are proximal to the cells. Figure 4B illustrates the same region of the microfluidic device as in Figure 4A at a later time point. The reporter molecules 412 can rapidly diffuse within the channel 122 and chambers 424, 426, 428 such that the concentration of the reporter molecules 412 equilibrate between the channel 122 and the interiors of the chambers 424, 426, 428. As illustrated in Figure 4B, the reporter molecules 4have reached a steady-state concentration to be substantially uniform within the chambers 424, 426, 428. The flow 242 of medium containing reporter molecule 412 is replaced by flow 242 of medium containing no reporter molecule 412, and the channel 122 does not contain significant amounts of reporter molecule. As the reporter molecules 412 within each chamber 424, 426, 428 contact the molecules of secreted analyte 410, the reporter molecules 412 can bind to the analyte 410, forming a reporter molecule:analyte complex 414, and providing a localized detectable signal that is related to the quantity of the secreted analyte 410. As flow 242 continues, reporter molecules diffuse out of the chamber, entering the channel 122 and are exported out of the microfluidic device. However, as shown in Figure 4B, diffusion of reporter molecule:analyte complex 410 is slower than that of unbound reporter molecule 412, due to its greater molecular weight (and effective size) and differentially does not diffuse as rapidly out of the chambers 424, 426, 428. Figure 4C illustrates the same region of the microfluidic device as in Figures 4A and 4B at yet a later time point at which the secreted analyte 410 and reporter molecule: analyte complexes 414 are diffusing from the source of the secreted analyte 410 (e.g., cells 402, 404, 406) to the channel 122. Flow 242 continues within the channel 122, thereby permitting reporter molecule 412 to diffuse out of each chamber 424, 426, 428 more rapidly than reporter molecule:analyte complex 410. The reporter molecule: analyte complex diffuses more slowly because secreted analyte molecules 410 may have a greater molecular weight (and associated effective size in solution) than the reporter molecules 412. In embodiments where the secreted analyte is an antibody and the reporter molecule is a peptide or aptamer, the difference in molecular weight is significant. In any case, the weight (and accordingly, the size) of the bound reporter molecule:analyte complex 414 is greater than that of the unbound reporter molecule 412 and, therefore, the reporter molecule:analyte complex 414 can diffuse more slowly than the unbound reporter molecule 412, providing a distinct diffusion profile and associated detectable signal, relative to the uniform signal provided by the unbound reporter molecules 412. Additionally, the biological micro-objects 502, 504, 506 continue to secrete the analyte 410, providing more targets for binding with reporter molecules 412 which are still disposed within the chambers 524,526,528. A time point can be selected where the percentage of unbound reporter molecules diffusing or already diffused out of the chamber exceeds a threshold value, permitting imaging of detectable signal from substantially or predominately only reporter molecule:analyte complex 414 within each chamber 424, 426, 428. In some embodiments, an assay image is acquired when the amount of unbound reporter molecules 412 that have diffused out of the chamber is about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or any range defined by two of the foregoing values. Alternatively, or in addition, in some embodiments an assay image is acquired when the amount of unbound reporter molecules 412 that have diffused out of the chamber is about 1.25X, 1.5X, 2.0X, 2.5X, 3.0X, 3.5X, 4.0X, 4.5X, 5.0X, 7.5X, 10X, 25X, 50X, or 100X greater than an amount of bound reporter molecule:analyte complexes 414 that have diffused out of the chamber The detectable signals obtained in the assay image may be proportional to the number of biological micro-objects in the chambers. Chamber 424 is illustrated as containing 6 biological micro-objects, chamber 426 is illustrated as containing 4 biological micro-objects and chamber 428 is illustrated as containing 2 biological micro-objects, and in some embodiments, the assay signal from the respective chambers may be proportional to those numbers of cells. In some embodiments, secretion of the analyte may depend on the cell cycle state at the assay signal acquisition time, and the signal from each of a plurality of chambers may not be substantially proportional to the number of cells within each chamber. Additionally, as illustrated in Figure 4C, while all of the cells 402, 404, 406 are secreting the analyte 410, different populations of cells (e.g., different clones) may secrete the biological analyte 410 at varying rates. Therefore the amount of analyte 410 produced (and the resulting intensity of the diffusion profile signal detected from the reporter molecule:analyte complex 414) may not be the same, chamber to chamber, even when normalized for the number of cells 402, 404, 406 present in each chamber. One or more assay images may be obtained during this period of time, which may be used to characterize the amount of secreted analyte 410 within the imaged chambers 424, 426, 428. Description of the analysis performed to arrive at relative or absolute quantification of the amount of secreted analyte 410 produced follows below. As stated above, the reporter molecule:secreted analyte (RMSA) complex diffuses more slowly because secreted analyte molecules may have a greater molecular weight (and associated effective size in solution) than the reporter molecules. In embodiments where the secreted analyte is an antibody and the reporter molecule is a peptide or aptamer, the difference in molecular weight is significant. In any case, the weight (and accordingly, the size) of the bound RSMA complex is greater than that of the unbound reporter molecule and, therefore, the RSMA complex can diffuse more slowly than the unbound reporter molecule, providing a distinct diffusion profile and associated detectable signal, relative to the uniform signal provided by the unbound reporter molecules.

The keying in on molecular weight differences between unbound reporter and RMSA complex may impact the compatibility of some assay methods discussed herein for workflows that involve the production of small molecular weight molecules (secreted analytes of small molecular weight). Since rate of diffusion is naturally a product of weight/size of the component in question, and distinct diffusion profiles allows for valuable detectable signals to be gathered, it is seemingly not apparent how to slow the diffusion of an assay reagent when binding a small molecular weight molecule that is secreted by cells. Hence, this may result in no discernable difference in diffusion rate between the bound and free fluorescent species, and thus seemingly no clear path to obtain the necessary diffusion profiles to gather the necessary detectable signals. By overcoming this problem, the various embodiments disclosed herein can be implemented in relation to secretion of, for example, small metabolites, biofuels, or other small molecules-of-interest. Measuring the production of small molecules can be accomplished, for example, by inverting the cell line development assay in accordance with various embodiments, wherein the brighter fluorescing chambers will indicate not the highest producing cell lines, but rather the weaker producing lines. In one example, a molecule (e.g., large molecular weight protein or protein complex, referred to as an "anchor") that is known to bind to small molecule targets, will first be bound off-chip with a fluorescent version of the target-of-interest. This fluorescent complex (anchor + fluorescent target) will be equilibrated throughout the chip, including within the chambers containing cells that could be secreting non-fluorescence versions of the target. In the presence of these unlabeled targets, binding kinetics will drive the exchange of the labeled targets with unlabeled targets at much higher concentration within the chambers. After flushing the channels clear for some time, the fluorescent targets not bound by an anchor will diffuse rapidly from the chamber. As a result, when reading fluorescence levels subsequent to this flush, the dimmer observed chambers will equate to those secreting more small molecular weight target. Figures 33A-C illustrate an example of this method, in accordance with various embodiments. In Figure 33A, high-MW anchor protein 3710 (e.g., antibody) with bound fluorescent molecule 3720 (labeled target) is introduced to a chamber 3750 containing cells 3760 and secreted unlabeled target 3730. In Figure 33B, binding kinetics drive the exchange of the labeled targets 37with unlabeled targets 3730. Assume, for example, the desire is to find cells producing the highest amount of FcIII peptide. To accomplish this, in view of above, one could add labeled FcIII pre-bound to an anchor off chip. After introduction of this labeled target into chambers, over time, an exchange in chambers between unlabeled FcIII secreted by cells and labeled FcIII pre-equilibrated onto anchor will occur. Finally, in Figure 33C, exchange is complete and channels can be flushed to allow unbound fluorescent targets to diffuse out of pen. From there, standard image capture and analysis (as discussed herein) can be used to observe fluorescence levels. However, as opposed to seeking brighter chambers as indicative of high secretion levels, since unbound fluorescent targets are removed, the dimmer chambers secrete more small molecule target. Diffusion assay under non-flow conditions in the microfluidic channel . Figures 5A through 5C illustrate an assay according to one embodiment of the disclosure. In Figure 5A, reporter molecules 412, each having a detectable label, are introduced into the microfluidic channel 122 of microfluidic device 500 by flowing a fluid containing a concentration of the reporter molecules 4into the channel 122. Figure 5A also shows chambers 524, 526, 528 fluidically connected to the microfluidic channel 122 containing various numbers of cells 502, 504, 506 secreting a biological analyte 410. Each of chambers 524, 526, 528 include a connection region 536 and an isolation region 540 (chamber 524 is the only pen labeled, just for clarity). The connection region 536 and isolation region 540 have properties as described above, and limit the contact of materials introduced into the channel 122 (e.g, within isolation region 540, materials flowing within the channel 122 may enter the isolation region only by diffusion, not by flow directly into the isolation region.) At the time point illustrated in Figure 5A, the molecules of the secreted analyte 410 are proximal to the cells. Figure 5B illustrates the same region of the microfluidic device as in Figure 5A at a later time point. The reporter molecules 412 can rapidly diffuse within the channel 122 and chambers 524, 526, 528 such that the concentration of the reporter molecules 412 equilibrate between the channel 122 and the interiors of the chambers 524, 526, 528. As illustrated in Figure 5B, the reporter molecules 4have reached a steady-state concentration equilibrium such that the concentration of the unbound reporter molecules 412 can be substantially uniform in the chambers 524, 526, 528 and the channel 122. The flow in the channel is stopped when the concentration of reporter molecules 412 is equilibrated into the chambers 524, 526, 528. As the reporter molecules 412 contact the molecules of secreted analyte 410, the reporter molecules 412 can bind to the analyte 410, forming a reporter molecule:analyte complex 414, and providing a localized detectable signal that is related to the quantity of the secreted analyte 410. Figure 5C illustrates the same region of the microfluidic device as in Figures 5A and 5B at yet a later time point at which the secreted analyte 410 and reporter molecule: analyte complexes 4are diffusing from the source of the secreted analyte 410 (e.g., cells 502, 504, 506) to the channel 122. There is no flow in the channel 122 at this time point. As above, the secreted analyte molecules 410 may have a greater molecular weight (and associated effective size in solution) than the reporter molecules 412. Therefore, the reporter molecule:analyte complex 414 can diffuse more slowly than the unbound reporter molecule 412, providing a distinct diffusion profile and associated detectable signal, relative to the uniform signal provided by the unbound reporter molecules 412. Additionally, the biological micro-objects 502, 504, 506 continue to secrete the analyte 410, providing more targets for binding with reporter molecules 412 which are still disposed within the chambers 524,526,528. The diffusion profiles and/or associated signals may be proportional to the number of biological micro-objects in the chambers. Chamber 524 is illustrated as containing 6 biological micro-objects, chamber 526 is illustrated as containing 4 biological micro-objects and t chamber 5is illustrated as containing 2 biological micro-objects. , In some other embodiments, however, the cells 502, 504, 506 in respective chambers 524, 526, 528 may secrete the analyte 410 at about the same rate, and the resulting intensity of detected signal from the reporter molecule:analyte complexes 414 may be proportional to the number of cells 502, 504, 506 present in each chamber. However, secretion of the analyte may depend on the cell cycle state at the assay signal acquisition time. Further, as illustrated in Figure 5C, while all of the cells 502, 504, 506 are secreting the analyte, different populations of cells (e.g., different clones) may secrete the biological analyte 410 at varying rates. Therefore the amount of analyte 410 produced (and the resulting intensity of the diffusion profile signal detected from the reporter molecule:analyte complex 414) may not be the same, chamber to chamber, even when normalized for the number of cells 502, 504, 506 present in each chamber. One or more assay images may be obtained during this period of time, which may be used to characterize the amount of secreted analyte 410 within the imaged chambers 524, 526, 528. Description of the analysis performed to arrive at relative or absolute quantification of the amount of secreted analyte 410 produced follows below. Secreted analytes. An analyte secreted by a biological micro-object may be a protein, a saccharide, a nucleic acid, an organic molecule having a molecular weight of less than 3Kd, a vesicle, a virus, and any combination thereof. A secreted analyte may be a naturally expressed analyte (e.g., natively expressed) or may be a bioengineered analyte (e.g., a product resulting from gene insertion, deletion, modification and the like). A secreted analyte that is a nucleic acid may be a ribonucleic or a deoxynucleic acid, may include natural or unnatural nucleotides. A secreted analyte that is a virus may be a viral particle, a vector or a phage. A secreted analyte that is a saccharide may be a mono-, di- or polysaccharide. Non-limiting examples of saccharides may include glucose, trehalose, mannose, arabinose, fructose, ribose, xanthan or chitosan. A secreted small, organic molecule may include but is not limited to biofuels, oils, polymers, or pharmaceutics such as macrolide antibiotics. A secreted analyte that is a protein can be an antibody or fragment of an antibody. A secreted analyte that is a protein can be a blood protein, such as an albumin, a globulin (e.g., alpha2-macroglobulin, gamma globulin, beta-2 microglobulin, haptoglobulin), a complement protein (e.g., component 3 or 4), transferrin, prothrombin, alpha 1 antitrypsin, and the like; a hormone, such as insulin, glucagon, somatostatin, growth hormone, growth factors (e.g., FGF, HGF, NGF, EGF, PDGF, TGF, Erythropoietin, IGF, TNF), follicle stimulating hormone, luteinizing hormone, leptin, and the like; a fibrous protein, such as a silk or an extracellular matrix protein (e.g., a fibronectin, laminin, collagen, elastin, vitronectin, tenascin, versican, bone sialoprotein); an enzyme, such as a metalloprotease (e.g., matrix metalloproteinase (MMP)) or other type of protease (e.g., serine protease, cysteine protease, threonine protease, aspartic protease, glutamic protease, asparagine peptide lyase), an amylase, a cellulase, a catalase, a pectinase, and the like; a bacterial, yeast, or protozoan protein; a plant protein; o or a viral protein, such as a capsid or envelope protein. A secreted analyte that is a protein can be an antibody, fragment of an antibody, an enzyme (including but not limited to a proteolytic enzyme), an engineered (normally intracellular protein) protein, such as for example, albumin, and/or a structural protein including but not limited to silkworm silk or spider silk). This list is not limiting and any protein that may be engineered to be secreted may be evaluated by the methods. The secreted analyte may be an antibody-drug conjugate. A non-limiting example of a secreted analyte that may have a combination of a protein, a saccharide, a nucleic acid, an organic molecule having a molecular weight of less than 3Kd, and/or a virus, can include a proteoglycan or glycoprotein. Reporter molecules and their characteristics. A reporter molecule may include a binding component designed to bind the secreted analyte and also may include a detectable label. The binding component may be any suitable binding partner configured to bind the secreted analyte. The binding component may be a protein, a peptide, a nucleic acid or small organic molecule having a molecular weight less than 3Kd. For example, the binding component can be a nucleic acid sequence that specifically binds another nucleic acid sequence or a peptide that specifically binds a protein (e.g. an epitope that recognizes a specific antibody). In some embodiments, the binding component can non-specifically bind a family of secreted analytes of a biological micro-object. For example, the binding component can be a peptide that specifically binds to an IgG domain or a nucleic acid that binds to a domain present in a family of nucleic acid sequences. In some embodiments, the reporter molecule may be multi-valent, comprising more than one binding component to bind more than one copy of the secreted analyte or to more than one member of a family of secreted analytes. For ease of discussion, the term secreted analyte as used herein can refer to either a specific secreted analyte molecule or a family of secreted analytes. The stoichiometry of the RMSA complex can therefore vary. For example, a reporter molecule binding one copy of the secreted analyte may have a RMSA complex with a 1:1 stoichiometry. Alternatively, the RMSA complex may have a 2:1, 3:1, 4:1, 2:2, 4:2, or other stoichiometry of the reporter molecule: secreted analyte. The reporter molecule may have any suitable molecular weight, with the provision that the apparent "size", as defined by diffusion characteristics of the reporter molecule: analyte complex which depend upon molecular weight, is sufficiently "larger" than the reporter molecule itself to observe differential diffusion between unbound reporter molecules and the RMSA complexes. The reporter molecule may have a molecular weight that is about 10 %, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or the same as the molecular weight of the secreted analyte. In some embodiments, the molecular weight of the reporter molecule is less than about 50%, 40%, 30%, 20%, 10% of the molecular weight of the secreted analyte. The molecular weight of the RMSA complex may be at least 2X, 4X, 10X, 20X, 30X, 40X, 50X or any number therebetween, greater than the molecular weight of the reporter molecule. The molecular weight of the RMSA complex may be 2-fold, 4-fold or 50-fold greater than the molecular weight of the unbound reporter molecule. Reporter molecules for one class of secreted analytes: antibodies. Reporter molecules suitable for binding to antibodies include proteins, peptides and aptamers configured to bind regions of an IgG. A non-limiting list of binding components suitable for use within a reporter molecule to detect an antibody is shown in Table 1. Table 1. Compounds as binding components of a reporter molecule to detect antibodies.CPD Affinity for IgG Molecular Weight (MW) Identification nanomolar 42kDa Protein A-AF594, PierceTM Recombinant Protein A (ThermoFisher Cat. # 77674) nanomolar 65kDa Protein G- AF594, PierceTM Recombinant Protein G (ThermoFisher Cat. # 21193) NA (Fc) Approx. 2.4kDa SEQ ID NO. 4 100nM (Fc) Approx. 2.4 kDa SEQ ID NO. 10 75 nm (Fc of hIgG) Approx .8 kDa SEQ ID NO. Aptamer-AF594, Apta-IndexTM (Apt.8, ID#44, Aptagen, LLC.) 8.6 nm (Fc) Approx. 12kDa Aptamer IgG Fc C02 #369 (Base Pair Technologies ATW007 NA, (Fc) ~2kDa to about 4.5 kDa SEQ ID. NO. 8 NA, (Fc) ~2kDa to about 4.5 kDa SEQ ID. NO. 9 NA, (Fc) ~2kDa to about 4.5 kDa SEQ ID. NO. 10 NA, (Fc) 2kDa to about 4.5 kDa SEQ ID. NO. 11 NA, (Fc) ~ 2kDa to about 2.4 kDa SEQ ID. NO. 12 NA, (Fc) ~ 2kDa to about 2.4 kDa SEQ ID. NO. 13 NA, (Fc) ~ 2kDa to about 2.4 kDa SEQ ID. NO. 14 NA, (Fc) ~2.4 kDa SEQ ID. NO. 15 Any of CPDs 1-14 can be used in the assays described herein. Some of the above listed CPDs are small peptides which are known to bind to the Fc domain of IgG (For CPD 4 and 7-14, see DeLano WL, et al. (2000), Science 287:1279-1283, and U.S Patent No. 7608681B2, the disclosure of each of which is incorporated herein by reference in its entirety). CPD 3 has a structure of Asp Ser Ala Trp His Leu Gly Glu Leu Val Trp Cys Thr (SEQ ID NO: 9). CPD4 has a structure of Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Thr (SEQ ID NO: 10). CPD 7 has a structure of Xaa 1-Xaa 2-Xaa 3-Xaa 4-Xaa 5-Xaa 6-Xaa 7-Xaa 8-Xaa 9-Xaa 10-Xaa 11-Leu-Val-Trp-Cys-Xaa 16-Xaa 17-Xaa 18-Xaa 19-Xaa 20, (SEQ ID NO: 1), where: Xaa 1 is any amino acid or absent; Xaa 2 is any amino acid or absent; Xaa 3 is any amino acid or absent; Xaa 4 is any amino acid or absent; Xaa 5 is Cys or Ser; Xaa 6 is any amino acid; Xaa 7 is any amino acid; Xaa 8 is any amino acid; Xaa 9 is any amino acid; Xaa 10 is any amino acid; Xaa 11 is any amino acid; Xaa 16 is any amino acid or absent; Xaa 17 is any amino acid or absent; Xaa 18 is any amino acid or absent; Xaa 19 is any amino acid or absent; and Xaa 20 is any amino acid or absent. CPD 8 has a structure of Xaa 1-Xaa 2-Xaa 3-Xaa 4-Xaa 5-Xaa 6-Xaa 7-Xaa 8-Xaa 9-Gly-Glu-Leu-Val-Trp-Cys-Xaa 16-Xaa 17-Xaa 18-Xaa 19-Xaa 20, (SEQ ID NO: 2), where: Xaa 1 is any amino acid or absent; Xaa 2 is any amino acid or absent; Xaa 3 is any amino acid or absent; Xaa 4 is any amino acid or absent; Xaa 5 is Cys or Ser; Xaa 6 is any amino acid; Xaa 7 is any amino acid; Xaa 8 is any amino acid; Xaa 9 is any amino acid; Xaa 16 is any amino acid or absent; Xaa 17 is any amino acid or absent; Xaa 18 is any amino acid or absent; Xaa 19 is any amino acid or absent; and Xaa 20 is any amino acid or absent. CPD 9 has a structure of Xaa 1-Xaa 2-Xaa 3-Xaa 4-Xaa 5-Xaa 6-Xaa 7-Xaa 8-Xaa 9-Gly-Glu-Leu-Val-Trp-Cys-Xaa 16-Xaa 17-Xaa 18-Xaa 19-Xaa 20, (SEQ ID NO: 3), where: Xaa 1 is any amino acid or absent; Xaa 2 is any amino acid or absent; Xaa 3 is any amino acid or absent; Xaa 4 is any amino acid or absent; Xaa 5 is Cys or Ser; Xaa 6 is Ala, Ser, or Thr; Xaa 7 is Trp or Tyr; Xaa 8 is His or Trp; Xaa 9 is Leu or Met; Xaa 16 is any amino acid or absent; Xaa 17 is any amino acid or absent; Xaa 18 is any amino acid or absent; Xaa 19 is any amino acid or absent; and Xaa 20 is any amino acid or absent. CPD 10 has a structure of Xaa 1-Xaa 2-Xaa 3-Xaa 4-Xaa 5-Xaa 6-Xaa 7-Xaa 8-Xaa 9-Gly-Glu-Leu-Val-Trp-Cys-Xaa 16-Xaa 17-Xaa 18-Xaa 19-Xaa 20, (SEQ ID NO: 4), where: Xaa 1 is any amino acid or absent; Xaa 2 is any amino acid or absent; Xaa 3 is any amino acid or absent; Xaa 4 is Ser, Arg, or Asp; Xaa 5 is Cys or Ser; Xaa 6 is Ala, Ser, or Thr; Xaa 7 is Trp or Tyr; Xaa 8 is His or Trp; Xaa 9 is Leu or Met; Xaa 16 is Glu, Ser, Thr, or Val; Xaa 17 is any amino acid or absent; Xaa 18 is any amino acid or absent; Xaa 19 is any amino acid or absent; and Xaa 20 is any amino acid or absent.

CPD 11 has a structure of Xaa 1-Xaa 2-Xaa 3-Xaa 4-Xaa 5-Xaa 6- Xaa 7-Xaa 8-Leu-Val-Trp-Cys-Xaa 13, (SEQ ID NO: 5), where: Xaa 1 is any amino acid or absent; Xaa 2 is Cys or Ser; Xaa 3 is any amino acid; Xaa 4 is any amino acid; Xaa 5 is any amino acid; Xaa 6 is any amino acid; Xaa 7 is any amino acid; Xaa 8 is any amino acid; and Xaa 13 is any amino acid or absent. CPD 12 has a structure of Xaa 1-Xaa 2-Xaa 3-Xaa 4-Xaa 5-Xaa 6-Gly-Glu-Leu-Val-Trp-Cys-Xaa 13, (SEQ ID NO: 6), where: Xaa 1 is any amino acid or absent; Xaa 2 is Cys or Ser; Xaa 3 is any amino acid; Xaa 4 is any amino acid; Xaa 5 is any amino acid; Xaa 6 is any amino acid; and Xaa 13 is any amino acid or absent. CPD 13 has a structure of Xaa 1-Xaa 2-Xaa 3-Xaa 4-Xaa 5-Xaa 6-Gly-Glu-Leu-Val-Trp-Cys-Xaa 13, (SEQ ID NO: 7), where: Xaa 1 is any amino acid or absent; Xaa 2 is Cys or Ser; Xaa 3 is Ala, Ser, or Thr; Xaa 4 is Trp or Tyr; Xaa 5 is His or Trp; Xaa 6 is Leu or Met; and Xaa 13 is any amino acid or absent. CPD 14 has a structure of Xaa 1-Xaa 2-Xaa 3-Xaa 4-Xaa 5-Xaa 6-Gly-Glu-Leu-Val-Trp-Cys-Xaa 13, (SEQ ID NO: 8), where: Xaa 1 is Ser, Arg, or Asp; Xaa 2 is Cys or Ser; Xaa 3 is Ala, Ser, or Thr; Xaa 4 is Trp or Tyr; Xaa 5 is His or Trp; Xaa 6 is Leu or Met; and Xaa 13 is Glu, Ser, Thr, or Val. The binding component is not limited to a material having a high affinity (e.g., nanomolar as is known for CPD 1 and CPD 2 of Table 1) for IgG. In some embodiments, binding components having affinities greater than about 100 millimolar or 1 micromolar may be successfully used in this diffusion based assay to detect antibodies. For other types of secreted analytes, different types of binding components of reporter molecules may be used. For example, an irreversible protease inhibitor may be used to detect a proteolytic enzyme, such as a fluoromethyl ketone inhibitor for serine or cysteine proteases. Aptamers to engineered analytes such as saccharides or macrolide antibiotics may be used. Antibodies or fragments thereof may be used to detect albumins, structural proteins, or macrolide antibiotics. Any suitable binding component to a secreted analyte may be used as is known in the art. Reporter molecule: purification tags . Current cell line development assays can depend on the presence of an Fc region common to antibodies. However, many therapeutic proteins and proteins produced for synthetic biology applications lack this moiety and are therefore may be incompatible with certain cell line development assays. Many of these non-antibody proteins either already include or could be designed to include genetically encoded residues that introduce short amino acids "tags" that are used for purification. In accordance with various embodiments, current on-chip titer assays could be modified to use fluorescently labeled small molecules that are specific to purification tags (i.e., short amino acids "tags" that are used for purification). This modification allows for solutions to large classes of proteins that are currently inaccessible to many cell line development assays. In one example, a hexahistidine (HIS 6) tag is encoded into the N- or C-terminus of a protein of interest; this tag is subsequently used to purify the HIS 6-labeled protein using a Ni-NTA (Nα,Nα-bis(carboxymethyl)-L-lysine, Nickel(II) complex) column, which specifically binds hexahistidine with high affinity. This chemical interaction can be leveraged to label the HIS6 tag with a fluorescently labeled species, such as Ni-NTA-Atto conjugates from, for example, Sigma. As is well known in the industry, Ni-NTA-Atto conjugates can provide specific and highly sensitive detection of HIS-tagged fusion proteins. The Ni-NTA-Atto complex, conjugated to Atto dye, is specific for polyhistidine tags and can perform with minimal cross-reactivity. Specifically, each Ni-NTA moiety binds to a pair of histidine side chains and each Ni-NTA can be labeled with a fluorophore. Moreover, two or three Ni-NTA moieties together can be cross-linked together to increase binding affinity. Reporter complex for low molecular weight secretions. As discussed above, measuring the production of small molecules (low molecular weight secretions) can be accomplished, for example, by inverting the cell line development assay in accordance with various embodiments, wherein the brighter fluorescing chambers will indicate not the highest producing cell lines, but rather the weaker producing lines. To provide for this, a molecule (e.g., large molecular weight protein or protein complex, referred to as an "anchor") that is known to bind to small molecule targets, can first be bound off-chip with a fluorescent version of the target-of-interest. This fluorescent reporter complex (anchor + fluorescent target) can be equilibrated throughout the chip, including within the chambers containing cells that could be secreting non-fluorescence versions of the target. In the presence of these unlabeled targets, binding kinetics will drive the exchange of the labeled targets with unlabeled targets at much higher concentration within the chambers. After flushing the channels clear for some time, the fluorescent targets not bound by an anchor will diffuse rapidly from the chamber. As a result, when reading fluorescence levels subsequent to this flush, the dimmer observed chambers will equate to those secreting more small molecular weight target. Detectable label. The reporter molecule may also include a visible, luminescent, phosphorescent, or fluorescent detectable label. In some embodiments, the detectable label may be a fluorescent label. Any suitable fluorescent label may be used, including but not limited to fluorescein, rhodamine, cyanine, phenanthrene or any other class of fluorescent dye label. Some examples of useful fluorescent dye labels include fluorescein (available as a thioisocyanate active species for labelling of the binding component of the reporter molecule) Alexa Fluor® 594 ((AF594, ThermoFisher Scientific, Cat. No. A20004 (NHS ester)) MW 819.8, Ex/Em590/617 nm) or HiLyte FluorTM 555 (AnaSpec Inc., Cat. # AS-81250) MW 869, Ex/Em 550/566 nm (Cy3 filter). In some embodiments, the reporter molecule, such as an aptamer or capture oligonucleotide, may include a FRET labeled oligonucleotide, which may include but is not limited to a molecular beacon, dual hybridization probe, Scorpion®, or Eclipse® probe. A FRET labeled oligonucleotide probe or probe pair may include fluorescent labels that do not fluoresce until a hybridization event takes place. In some embodiments, the detectable label is covalently attached directly or indirectly to the binding component of the reporter molecule. In some other embodiments, a capture oligonucleotide may be a binding component of a reporter molecule and either an intrinsic or extrinsic fluorescent dye may be the detectable label, such that the detectable label of the reporter molecule may not be detectable until the capture oligonucleotide binds the analyte, for example, an intercalating dye. In some embodiments, a detectable label of a reporter molecule may not be detectable until after the RMSA complex has formed, as the detectable signal is shifted to a new wavelength not present prior to binding. In some embodiments, such as an intercalating dye covalently attached to the binding component of the reporter molecule. In other embodiments, the detectable label may be an isotope. In yet other embodiments, the detectable label and the binding component is a single moiety, for example a protein or nucleic acid that provides a detectable signal (e.g. a self-detectable protein such as a green fluorescent protein (GFP), or a ribonucleic acid aptamer such as "Spinach", which is an RNA equivalent to GFP. Spinach incorporates 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) as the fluorescent detectable label. Diffusion modelling. The methods described herein utilize models and observations related to differential diffusion of secreted analytes from the isolation region of a chamber to the flow region (e.g., microfluidic channel). A number of software programs may be used in modelling the behaviors of secreted analytes of a biological micro-object including, but not limited to COMSOL®, MATLAB® and/or various numerical modeling and computer-assisted design tools. Figure 6 shows a model of one type of chamber having one biological cell (602) placed at the base of the chamber 624, within the isolation region 640, at a point distal to the opening of the chamber 624 to the channel 122. Lines of diffusion 610 show the trajectory of diffusion of a secreted analyte of the cell 602 from the isolation region 640 through the connection region 636 to the channel 122. It can be seen that as diffusing materials passage through the connection region the lines of diffusion concentrated and flow linearly to the channel 122. The rate of diffusion is defined by the secreted analyte’s coefficient of diffusion and can be modeled as follows. The diffusion coefficient, D for a specific secreted analyte is defined as: D = (1/f) kT (Equation 1) where f is a frictional coefficient, k is the Boltzman constant, and T is the absolute temperature. The frictional coefficient f is dependent upon the viscosity ( of the solvent in which the secreted analyte is diffusing and on the size and shape of the secreted analyte. A secreted analyte having a spherical sphere has a minimalized frictional coefficient, but a nonsymmetrical shape such as that of an antibody or other protein with defined structural constraints will result in larger f. Additionally, if the secreted analyte has interactions with the solvent such as hydrogen bonding or waters of hydration associated with the secreted analyte, the frictional coefficient will also be increased. Some genericized diffusion coefficients are shown in Table 2. Table 2. Exemplary diffusion coefficients.Generic material Diffusion Coefficient Small molecule (< 1kDa) in water 1-1.5 x 10-5 cm s-1 Small protein ( < 20kDa 10-6 cm s-1 Diffusion of the secreted analyte can be represented by the following equation: = q iDt (Equation 2) where is the mean squared displacement, and x is the mean distance from a selected starting point of travel over time t. The value of q i depends on whether diffusion is being evaluated in 1, 2, or 3 dimensions. With these equations, the time for a reporter molecule to diffuse in and out of a chamber of defined configuration and the time for the RMSA complex can be modelled, and is shown in Figures 7A and 7B for an antibody secreted analyte, having a molecular weight in the range of about 1kDa. In Figure 7B, curve 712 models the behavior of a small peptide like CPD 3, molecular weight of about 2.5kDa, where the small peptide is calculated to be capable of diffusing into and equilibrating within a chamber configured like the chamber 424, 524, 624 and the like (Figures 4A-C, and 6 respectively) from the channel in within 25-30 min. In contrast, curve 714 models the behavior of the much larger CPD 1, having a molecular weight of about 45 kDa. This larger molecule presents more opportunities to interact with the solvent, and full equilibration is not reached somewhere between 45-50 min. In Figure 7B, the behavior of four different species is shown for diffusion out of a chamber configured like that of 424, 624. In this graph, curve 716 shows the calculated rate of diffusion out of a chamber configured like that of chamber 424, 524, 624, and the like, for the small peptide CPD 3. Small peptide CPD 3 is substantially eliminated from the chamber by about 25 min. In contrast, when CPD 3 is bound to the secreted analyte IgG (MS 150 kDa), curve 722 (triangular shape) shows the calculated diffusion behavior for the RMSA complex containing CPD 3, where a small amount of RMSA complex remains after 60 min. Curve 718 (diamond shape) shows the calculated diffusion behavior for CPD 1 (protein A, a 45kDa protein), which accordingly requires over 50 min to diffuse out substantially completely. When this protein complexes with the secreted antibody (MW 150 kDa) curve 724 (dashed segment) shows a similarly slow rate of diffusion to that of CPD 3: IgG complex, and still shows complex remaining after 60 min. Figure 8A shows the calculation of the difference between reporter molecule and RMSA complexes for each of the two different binding components of the reporter molecule. Curve 8shows the assay time optimization for the maximal difference in concentration within the chamber for the CPD 3: CPD 3/IgG pairing, in order to observe the maximum signal arising from the RMSA complex and the minimal signal due to unbound reporter molecule, which appears to be at about 15 minutes of restored medium flow in the microfluidic channel, which exports any diffused material out of the microfluidic device. Curve 828 shows the difference curve for CPD 1, showing the difference in concentration between the unbound CDP-1 and RMSA complex containing CPD 1 within the chamber, and showing that the maximized difference is out at a later timepoint, sometime after minutes or longer. Figure 8B shows the experimental time for diffusion in (upper graph) and diffusion out (lower graph) for unbound CPD 1, showing reasonable correlation with the calculated values. These sets of modelling and executed experiments shows that it is possible to find optimized points in time for observation of a detectable signal from substantially the reporter molecule: secreted analyte complex in order to assess the levels of analyte secretion within a specific chamber. Selection of a region along an axis of diffusion. Figures 9A-B and 10A-B show modelling experiments used to determine a region from which to extract quantitative measurements (either relative or absolute) from assay images. In Figures 9A-B, modelling of diffusion flow and resultant fluorescent signal intensity from the RMSA complex was performed to consider the effect of location of biological cell 902, 904, 906 within chamber 924 of microfluidic device 900, which is similar to chamber 424, 524, 624. The effect was modeled using a location for cell 902 at about 25 microns from the base of the chamber (0 microns), which is distal to the opening of the chamber 924 to the channel 122; cell 904 was modeled at a distance about 100 microns from the base of the chamber 924; and cell 906 was modeled at a distance about 180 microns from the base of the chamber 924 (see horizontal axis of Figure 9A and Fig 9B). Each cell is modeled to lie along a center axis of diffusion trajectory towards the opening of the chamber, illustrated by line 952, for modelling simplicity. Each of these positions lie within the isolation region 940 of the chamber, and well away from the connection region 936 of the chamber, therefore insuring that signal intensity detected from cells 902, 904, 906 within the isolation region 940 are not affected by flow effects from flow 242 in the channel 122. The y axis of Figure 9A represents the normalized concentration of reporter molecule (or equivalently RMSA complex, as this experiment relies only upon fluorescence intensities detected.) As shown in Figure 9A, the intensity of the fluorescent signal (modelling includes the constraint that 902, 904, 906 are all producing secreted analyte at the same rate) is highest for cell 902, as the fluorescently labeled complex is diffusing in a more monodirectional manner than that of 904, 906, due to its location near the base of the chamber 924. Cells 904, 906 have more capacity to have labeled RMSA complexes diffusing in all directions. What is determined by this model is that a region can be identified, where the signal intensity is most sensitive to changes in fluorescent signal intensity due to changes in labeled species’ concentrations (e.g., RMSA complex), and least sensitive to the exact location of cell 902, 904, 906, which is region 944, which lies along the axis of diffusion between the chamber 924 and the channel 122, as indicated both in Figure 9A and 9B. Cell position insensitive region 944 is at least a portion of an area of interest (AOI) used to assess the relative or absolute amount of a secreted analyte of a biological micro-object within a chamber 924. In some embodiments, an AOI may include additional portions of the chamber 924 and/or the channel 122. Chamber optimized for large populations of cells.Figure 10A and 10B illustrates a similarly constructed modelling experiment as shown in Figures 9A and B for a differently configured chamber 1024. The effect of location of biological cell 1002, 1004, 1006 within chamber 1024 (which is similar to chamber 224, 226, 228) of microfluidic device 1000 is shown. The effect was modeled using a location for cell 1002 at about 25 microns from the base of the chamber (microns), which is distal to the opening of the chamber 1024 to the channel 122; cell 1004 was modeled at a distance about 100 microns from the base of the chamber 1024; and cell 1006 was modeled at a distance about 180 microns from the base of the chamber 1024 (see horizontal axis of Figure 10A and Fig 10B). Each cell is modeled to lie along a center axis of diffusion trajectory towards the opening of the chamber, illustrated by line 1052, for modelling simplicity. Each of these positions lie within the isolation region 1040 of the chamber, and well away from the connection region 1036 of the chamber, therefore insuring that signal intensity detected from cells 1002, 1004, 1006 within the isolation region 1040 are not affected by flow effects from flow 242 in the channel 122. The y axis of Figure 10A represents the normalized concentration of reporter molecule (or equivalently RMSA complex, as this experiment relies only upon fluorescence intensities detected), and the discussion of the concentrations shown are as above for Figure 9A. What is determined by this model is that a region can be identified, where the signal intensity is most sensitive to changes in fluorescent signal intensity due to changes in labeled species’ concentrations (e.g.,RMSA complex), and least sensitive to the exact location of cell 1002, 1004, 1006, which is region 1044, which lies along the axis of diffusion between the chamber 1024 and the channel 122, as indicated both in Figure 10A and 10B. Cell position insensitive region 1044 is at least a portion of an AOI used to assess the relative or absolute amount of a secreted analyte of a biological micro-object within a chamber 1024. In some embodiments, an AOI may include additional portions of the chamber 10and/or the channel 122 which are located along the axis of diffusion between the chamber 1024 and the channel. In some embodiments, the geometry of a chamber may be altered to provide an optimal diffusion profile of a secreted analyte. Figure 11A illustrates a section of a microfluidic device 11comprising a channel 122 and chambers 1124, 1126 designed to provide an optimized diffusion profile. Specifically, the chambers 1124, 1126 have isolation regions which can accommodate a large number of biological micro-objects 1102, which may be useful in providing a larger signal intensity for use in assessing the quantity (relative or absolute) of secreted analytes of the cells 1102. In some embodiments, the isolation region 1140 of chamber 1124 may accommodate a volume ranging from 0.1 to 100 nL. In a specific embodiment, as shown in Figure 11B, the isolation region1140 may hold a volume of 6 nL. The chambers 1124, 1126 can accommodate as many as 100, 200, 300, 400 or 500 micro-objects. In some embodiments, the chambers may accommodate a maximum of 300-400 micro-objects. The chambers 1124, 1126, each have a connection region 1136 that is configured to separate biological micro-objects 1102 in the isolation region 1140 from the connection region 1136, creating sufficient distance for the secreted analyte to diffuse away from its source (e.g., one of the biological micro-objects 1102 secreting the analyte). This separation reduces interference or overlap of localized signal from RMSAcomplexes still associated at or on the biological micro-obejct 1102 (e.g., not freely diffusing) with its diffusion trajectory along line of anticipated diffusion trajectory 1130. By eliminating this overlap, concentration values generated from the at least a portion of the AOI or the entire AOI will represent signal from the bound reporter molecule as it diffuses. In some embodiments, the connection region 1136 is separated from the isolation region 1140 by constriction of the connection region 1136 relative to the isolation region 1140. In some embodiments, the connection region 1136 will have a width ranging from 10-30 microns and a length ranging from to 200 microns. In a specific embodiment, the connection region 1136 is 20 microns in width and range from 100 to 200 microns in length. Figure 11B depicts the flux lines 1120 and concentration gradient lines 110 of secreted analyte of biological micro-object 1102 from within chamber 1124, through the connection region 1136 and out to channel 122. Portions of the connection region 1136 may be selected as at least a portion of an AOI and may be part of the region which is insensitive to cell location and is sensitive to variance in intensities observed in the Assay image.

Assessing an Area of Interest (AOI). Figure 12A shows a schematic representation of an AOI from which data is extracted for the determination of the relative or absolute amount of a secreted analyte from a biological micro-object. The AOI 1250 is selected to encompass: a region in the isolation region 1240, region in the connection region 1236 (of chamber 1224 in microfluidic device 1200); and a portion of the channel 122, all of which is aligned along the axis of diffusion from the chamber 1224 to the channel 122. In this embodiment, flow 242 is present in the microfluidic channel 122, reducing any detectable signal within the portion of the channel incorporated within the AOI. The selection of the point at which the AOI ends within the chamber is made to prevent overlap with the biological object 1202 which secretes the analyte, and from which detectable signal emanates. As shown in Figure 12A, lines of diffusion 1210 are directed towards the connection region 1236 and become aligned with the axis of diffusion as the connection region 12is entered. Concentration gradient lines 1220 are shown as well. Figure 12B is a photograph showing the Assay Image for a chamber 1224, having an identification number 1260 of "327", which indicates its location within the microfluidic device 1200. The identification number assists in correlating brightfield and fluorescence image locations, and also assists users to select, manipulate and export cells from a selected chamber. Chamber 1224 of Figure 12B has one biological micro-object 1202 present within the isolation region (not labelled). The Assay Image clearly shows extensive amount of fluorescence signal within the chamber, emanating from biological micro-object 1202. The AOI 1250 is shown photographically imposed, and is aligned along the axis of diffusion and centered along the line of diffusion trajectory 1252. The AOI is 20 pixels wide, which is chosen depending on the width of the connection region 126 (not labelled in Figure 12B) and is divided into 20 sub-regions. An AOI may have other pixel sizes to each sub-region and the number of sub- regions may vary from about 1 to about 50. The sub-region 1254 of the AOI is the sub-region located furthest away from the channel 122 of all the sub-regions of the AOI, but is selected to not overlap with the biological micro-object 1202. The sub-region at the second end of the AOI is sub-region 1258, which is located within the channel 122. Importantly, the group of sub-regions 1256 is the Cell position insensitive region 944, 1044 of Figures 9A-B and 10A-B, from which the detected fluorescence is used to assess the relative or absolute amount of a secreted analyte of a biological micro-object within a chamber. Figure 12C shows a graphical representation of the fluorescence detected in the AOI, where the values on the horizontal axis represent sub-regions 1 (corresponding to sub-region 1254 of Figure 12B), the sub-region at the most proximal end of the AOI to the biological micro-object 1202, and sub-region 20 corresponds to the sub-region 1258 of Figure 12B, at the most proximal end of the AOI in the channel 122. The amount of detected fluorescence in the AOI is proportional to the amount of secreted analyte. A variety of mathematical operations may be used to extract information about the relative or absolute amount of the secreted analyte and is discussed in detail in sections below. Normalization of the Assay image . Before the Assay Image can be processed to assess relative or absolute amounts of a secreted analyte, the raw Assay Image may be normalized. Figure 13A shows a raw Assay Image which displays error such variance and non-linearity of system components such as the light source(s), the Light Modulating Subsystem (e.g. the DMD), the image capture device (e.g. the camera). Method A for assays conducted under flow conditions. In one embodiment, the raw Assay Image may be normalized by subtracting both a Dark Reference image and a Signal Reference image correction from each pixel in the raw Assay Image as in the following equation: (Equation 3) The Dark Reference image may be obtained by imaging the microfluidic device before flowing any medium into the device. Autofluorescence errors and other system errors can be corrected by subtracting the Dark Reference value at each pixel. The Signal Reference Image may correct for roll off, photobleaching errors or camera errors, and is obtained by flowing reporter molecule, or just the reporter molecule throughout the microfluidic device to reach an equilibrated concentration of the reporter molecule or fluorescent label. Each pixel in the raw Assay Image may be corrected in this manner, before extracting the fluorescence data for quantitation purposes. The normalized Assay Image is shown in Figure 13B. Method B for some embodiments of assays conducted under non-flow conditions . As a first step in normalization, the Dark Reference image, as described above, was subtracted from the image of the microfluidic device with the bound and unbound reporter molecules present to produce an "dark reference subtracted image." As a second step, portions of the raw Assay image of Figure 13A where the bound and unbound reporter molecules are not present (i.e. walls defining the chambers and channels in the microfluidic device) were removed or "masked" from the auto-fluorescence subtracted image to produce a "masked dark reference subtracted image." As understood by those skilled in the art, this step also could be performed before the subtraction of auto-fluorescence. As a third step in generating the normalized image of Figure 13A, the intensity value for each pixel in the masked, auto-fluorescence subtracted image was divided by the global average intensity calculated based on all pixels in the masked, auto-fluorescence subtracted image. By dividing the intensity value for each pixel by the global average intensity, an image or similar data structure (e.g. a matrix) comprising a gain correction factor for each pixel is generated ("gain correction image") is for each pixel of the image is produced. Other methods of producing a gain correction image are well known to those skilled in the art. As a fourth step in generating the normalized image depicted in Figure 13A, the gain-correction image was subject to a smoothing algorithm to reduce random noise. This step may not be employed in some embodiments of the method. Specifically, the gain-correction image was subject to a box-filter smoothing algorithm that used a 9-pixel by 9-pixel box-filter that accounts for the masked portions of the image in generating a local average for each pixel. As can be appreciated by those skilled in the art, other smoothing algorithms such as mean filtering, Gaussian filtering, gradient weighting filtering, sequence statistical filtering, robust smoothing filtering, Crimmins noise removal filtering, edge preserved filtering and self-adaptive median filtering may be used. As a fifth step in generating the normalized photograph depicted in Figure 13B, the smoothed gain-correction image may be multiplied by the auto-fluorescence subtracted image to produce a normalized image. These methods may combine any of the foregoing steps and methods in the same or different sequence. Method C for some embodiments of assays conducted under non-flow conditions . Another method of normalizing the image may be used, depending on the substantially uniform concentration unbound reporter molecule within the channel due to its greater rate of diffusion over that of the bound RMSA complex. The brightness of the channels may be used to normalize the image to correct for the errors described above. Therefore, in an alternate embodiment, the normalized image of Figure 13B can be obtained using the brightness in the channels proximal to the chambers to correct for any variance in the amount of brightness across the view of regions of the microfluidic device. This method of normalization relies on the fact that the channels are not expected to have any analyte (or any RMSA complex) present and therefore can be performed using any area of the microfluidic device that does not have the analyte present. In order to normalize based on the channel intensity, as a first step, a region of the channel R that is not expected to have any analyte present in it is identified for each chamber. In some embodiments, this region R can be a pre-defined region R corresponding to an area of the channel above the chamber. In other embodiments, the region Rfor each chamber can be identified based on other information or calculated based on the image. For each region of the channel R , a brightness value B R is computed based on the pixels within the region. Prior to computing the brightness values, the image used to calculate the brightness value may be subtracted, masked or otherwise processed as discussed above. In some embodiments, B R is the average brightness value of the pixels within the region R . After the average brightness value B R for each region R is computed, the image of the chambers and channels may be partitioned into a series of areas A , where each area A encompasses a respective region R . This area may be computed so that a region R is in the center of an area A. In a specific embodiment, the areas A may be computed by generating a Voronoi diagram or a Delauney triangulation of the centroids of each region R. In other embodiments, each region R need not be centered in its respective area Aand can be computed based on pre-defined areas segmenting the microfluidic device. For each area A , a gain-correction factor is calculated based on the maximum brightness value calculated for the brightest region B Rmaxdivided by the brightness value B R for the region R associated with the area A . The gain-correction factor may be used to generate a gain-correction image which can be multiplied against another image (e.g. the auto-fluorescence subtracted image) to produce a normalized image. The gain-correction factor image may also be smoothed as described above prior to use in normalization. Quantification of the assay signal. In some embodiments, the diffusion profile of the RMSA may be used to quantify the amount of the RMSA complex present in the chamber. The diffusion profile provides a series of values ("concentration values") that represent the concentration of the RMSA complex as it diffuses from its source to the channel. After identification of the AOI, other transformations may be applied. For example, the pixels in each line may be processing by discarding outlier and/or aberrant pixels, other forms of global/local normalization, space conversion, and transforming the space of the pixel (e.g. from a multi-dimensional space to a two-dimensional space or vice-versa). Depending on the embodiment, the intensity values may be used in different ways to calculate the concentration values. In some embodiments, the AOI may be sampled at fixed points to generate a set of concentration values corresponding to the intensity values at the fixed points. In some embodiments, the AOI may be segmented in a series of segments and the median or mean intensity of each segment may be calculated. Based on the embodiment and the degree of resolution required, the number of concentration values calculated can be as low as 1 and as high as the number of pixels in the line representing the diffusion trajectory. Depending on the embodiment, the concentration values may be combined in different ways in order to quantify the amount of signal from the bound reporter molecule (and therefore the amount of secreted analyte) present. In some embodiments, the concentration values may be plotted to assess whether concentration values exhibit characteristics consistent with a diffusion profile. Depending on the embodiment, a number of algorithms may be used to fit a line to the concentration values and calculate characteristics of the line such as the slope and error associated with the line. Suitable line-fitting algorithms include: least-squares, polynomial fit, curve-fitting, and erfc fitting. Other algorithms are known to those skilled in the art. Methods of transforming fluorescence intensity values to obtain concentration values is described more fully below. Figure 14A is an Assay Image (photograph) of one chamber 1424, having an identification number "1107", and wherein a line of anticipated diffusion trajectory 1452 is shown. An AOI 1450 is projected onto the Assay Image, and in this example, has a width of about 12 pixels, and it was segmented into 20 equal segments along the axis defined by the line (segments not shown). The median intensity for each of the 20 equal segments was calculated and then plotted as the concentration value in the graph of Figure 14B. On the horizontal axis of the graphs, the segment numbers 1-20 are numbered according to their distance from the source (i.e. the cells secreting the secreted analyte), with the segment numbers having a low number representing the segment of the AOI closest to the cells in the region of the chamber most distal from the channel. Figure 14B depicts a series of curves representing concentration values for a set of chambers, which were generated according to the method discussed in the previous paragraph and other sections following. To generate the series of curves shown in Figure 14B, the concentration values generated for each chamber were not normalized based on the number of cells in the chamber. However, in alternate embodiments, the concentration values and resultant curves may be normalized based on the number of cells in each chamber. As shown in Figure 14B, the slope of the curve (of concentration values) for each chamber may be used to assess the relative amount of the secreted analyte present in each chamber. In other words, the slope may be used as a score such that chambers can be ranked and ordered relative to each other, and "slope" and "score", in some embodiments herein, may be used interchangeably. In some instances, the score may be referred to as a secretion score. More specifically, in instances where the secreted analyte is produced by a biological micro-object (e.g. cell) present in the chambers, the slopes may be used to assess the relative ability of the cells in each chamber to produce the secreted analyte (e.g. the relative ability of cells to secrete an antibody). As discussed below, a relative or absolute amount of the secreted analyte may be calculated using different methods, including summing all the points in the sub-region of the AOI which is insensitive to the positions of the cells in the chamber and is most sensitive to variance in fluorescence intensity observed (e.g., regions 1256 of Figure 12B, 1044 of Figures 10A-B, and 944 of Figures 9A-B). In addition, the shape of the curve may be evaluated to assess whether the concentration values for each chamber conform to expected parameters or indicate systemic error. For example, the shape of the curve labelled "Pen 1497" in Figure 14B does not correspond to the shape of the curves observed for the other chambers whereas the shape of the curve labelled "Pen 1107" does corresponds to the expected diffusion profile. As shown in Figure 14A, Pen 1107 had a visible gradient of reporter molecule from its chamber to the channel which resulted in its curve corresponding to an expected diffusion profile. As shown in Figure 14C, a chamber 1426, having identification no. Pen 1497, has a line of anticipated diffusion trajectory 1452and AOI 1450. However, chamber 1426 is proximal to a channel containing a bubble, where the meniscus 1401 of the bubble appears in the image as a white ellipse. The presence of the bubble results in the aberrant curve for Pen 14depicted in Figure 14B. In various embodiments, the region of the segmented AOI that linear regression may be applied may be selected to be segments (sub-regions) 9-13, which as discussed above encompass portions of the connection region and have been identified to be most sensitive to fluorescence intensity variance and most insensitive to the location of biological micro-objects within the chamber. Figure 15 shows an overlay of a plurality of curves representing intensity values (and thereby concentration values) derived via any of the methods described herein, obtained from a plurality of chambers within a microfluidic device. The intensity values of each point in each curve, plotted against the vertical axis of the graph, have been normalized for ease of overlay. The values along the horizontal axis start with a value of "y" equal to zero, representing the first pixel in the y dimension of each AOI (and is physically located within the channel of the microfluidic device and outside of the chamber, similarly to the AOIs shown in Figures 9A-B, 10A-B, 12A-b, and 14A and C. The points along the horizontal axis marked "200" correspond to the last pixel in each AOI of the plurality of chambers, which is the boundary of the AOI closest to the cells secreting analyte, and hence the source from which the detectable signal from RMSA complex emanates. The concentration values obtained from the portion 1544 of the AOI that is least sensitive to the position of cells within the chamber and most sensitive to the variance in fluorescence intensities is shown in the portion of the curve associated with y values between about 90 and about 130, as shown. It can be seen that a mathematical operation imposing a linear shape in this region, and extracting the slope thereof, closely represents the state of the data. Performing the assay across the nanofluidic device containing thousands of clonal populations, each derived from a single cell placed into a separate chamber, can provide quantification of each of the clonal populations. As shown in Figures 16A and B, the ability to find rare high producing clones is enhanced. If it is assumed that distribution of titers from a randomly secreting pool of cells is well described by Poisson statistics, then the titer distribution should fit to a gamma distribution. In Figure 16A, the curve superimposed over the bar graph distribution of titers (which are obtained from the scores and normalized for number of cells present in each chamber of the plurality, and expressed in Arbitrary Units (A.U.) shows good agreement. There is a great majority of clonal populations expressing analyte from less than 50 to less than 100 A.U, and very few individual titers out in the high range of 250 A. U. and over. The same data is now shown plotting the relative specific productivity against rate of growth (along the horizontal axis). The curves superimposed on the graph show lines of constant titer, which again show that the majority of clones whether they are fast or slow growing clones, express the analyte at less than 100 A.U. and are not the desirable highly producing clones sought for cell line development. Only a few clones identified within the regions 1670, 1680, and 1690 are the rare high producers. However, these clones are not the fastest producing clones arising out of the originally seeded single cells. If these cells were mixed in with other cells as part of a larger growth environment, such as a well plate or a shaker flask, these rare, highly producing clones would most likely be overgrown by the faster growing, less productive clones. Trying to identify these clones if one attempted selection of random single cell sets for expansion, would require a massive sampling effort with massive input of resources to grow up the number of cells that would be required to have the probability of seeing them. In the system provided here, the titer (or score), may be obtained for all of the clonal populations, and the physical location of the productive clones is known (see Figure 21, below). Further, only the selected clones may be selected and physically moved for further expansion/subcloning; selection and movement may be performed individually to prevent contamination by other cell populations. The opportunity to screen all of the clones arising from the originally seeded cells provides a greatly improved process for screening and selecting cells that secrete a desired analyte. In accordance with various embodiments, fluorescence recovery after photobleaching (FRAP) can be another technique for making on-chip concentration measurements (i.e., secretion rate) of secreted molecules. In particular, the concentration and/or binding affinity of an unlabeled molecule secreted from a cell should be detectable by monitoring the fluorescence recovery after photobleaching. The technique can be implemented across various points in various cell line development assays (CLD assay) discussed herein such as, for example, during an equilibration step that is generally performed to create a homogeneous reagent concentration across the chip. FRAP could be performed during this step, where in each chamber there is a population of free reagent, a population of secreted molecules, and a population of reagent bound to secreted molecule(s) of interest. By photobleaching an area of the chamber, such as, for example, a 50-100 um box, or even the neck itself, free reagent and bound populations re-diffusing into that area can be observed. The free reagent generally recovers quickly because of its fast diffusion rate, while the bound complex recovers slowly due to its slower diffusion rate. The ratio of the fast-recovery process magnitude (hin Figure 31) to the slow recovery process magnitude (h2 in Figure 31) could then form a measure of concentration (secretion rate), with recovery time constants being determined by the molecular weights of the reagent and bound complex respectively. Part A of Figure 31 represents a low secreting molecule while Part B of Figure 31 represents a high secreting molecule. Moreover, OptoElectroPositioning (OEP) technology, discussed in more detail below, could be used to get more optical power to decrease bleach time, allow for use of a less stable dye, and allow for imaging at lower projector power during a monitoring step. In accordance with various embodiments, therefore, a method of assessing a level of secretion of an analyte by a biological micro-object, or a population of biological micro-objects generated therefrom is provided. The method can comprise introducing the biological micro-object into a chamber of a microfluidic device, wherein the microfluidic device comprises an enclosure having a flow region, wherein the chamber is fluidically connected to the flow region, and wherein the chamber contains a first fluidic medium. The method can further include allowing the biological micro-object, or the population of biological micro-objects generated therefrom, to secrete an analyte into the first fluidic medium within the chamber, and introducing a second fluidic medium into the flow region for a first period of time, wherein the second fluidic medium comprises a plurality of reporter molecules. The method can further include allowing a portion of the plurality of reporter molecules to diffuse into the chamber and bind to the analyte secreted therein, thereby producing a plurality of reporter molecule: secreted analyte (RMSA) complexes, and detecting reporter molecules located within an area of interest within the microfluidic device, wherein the area of interest includes at least a portion of the chamber. The reporter molecule (or each reporter molecule) can comprise a binding component configured to bind the secreted analyte, and a detectable label. In accordance with various embodiments, a non-transitory computer-readable medium is provided in which a program is stored for causing a computer to direct a system to perform a method for determining a quantity of analyte produced by a biological micro-object, or a population of biological micro-objects generated therefrom, using FRAP techniques. An example computer system for this is provided by the block diagram of FIG. 25 illustrating a computer system 3100, upon which embodiments of the present teachings may be implemented. Details of computer system 3100 will be provided below. In accordance with various embodiments for assessing a level of secretion of an analyte by a biological micro-object, or a population of biological micro-objects generated therefrom, using FRAP techniques, the detectable label can comprises a visible, luminescent, phosphorescent, or fluorescent label. The detectable label of the reporter molecules can be a fluorescent label, wherein said detecting the reporter molecules comprises detecting fluorescence emission from the fluorescent label of the reporter molecules within the area of interest.

In accordance with various embodiments for assessing a level of secretion of an analyte by a biological micro-object, or a population of biological micro-objects generated therefrom, using FRAP techniques, methods can further comprise exposing, for a second period of time, a portion of the microfluidic device comprising the chamber to electromagnetic radiation comprising a wavelength which is capable of exciting the fluorescent label of the reporter molecules. Methods can further comprise detecting fluorescence emission within the area of interest is performed after the second period of time, detecting fluorescence emission within the area of interest is performed two or more times during a third period of time, and/or detecting fluorescence emission within the area of interest is performed substantially continuously during a third period of time. In accordance with various embodiments for assessing a level of secretion of an analyte by a biological micro-object, or a population of biological micro-objects generated therefrom, using FRAP techniques, methods can further include exposing, for a fourth period of time, a portion of the microfluidic device comprising at least a portion of the chamber to electromagnetic radiation but not the flow region, wherein the fourth period of time is sufficient to photobleach the fluorescent label of any reporter molecules present in the portion of the chamber, and detecting fluorescence emission within the photobleached portion of the chamber. The detecting fluorescence emission within the photobleached portion of the chamber can be performed after the fourth period of time, can be performed two or more times during a fifth period of time; and/or can be performed substantially continuously during a fifth period of time. Further, the detecting fluorescence emission within the photobleached portion of the chamber can occur about 5 seconds to about 20 seconds after said exposing for a fourth period of time. Moreover, the steps of exposing for a fourth period of time and detecting fluorescence emission in the photobleached portion of the chamber can be repeated one or more times. In accordance with various embodiments for assessing a level of secretion of an analyte by a biological micro-object, or a population of biological micro-objects generated therefrom, using FRAP techniques, the photobleached portion of the chamber is comprised by the area of interest. In accordance with various embodiments for assessing a level of secretion of an analyte by a biological micro-object, or a population of biological micro-objects generated therefrom, using FRAP techniques, the chamber has an isolation region and a connection region fluidically connecting the isolation region to the flow region, wherein the isolation region and the connection region are configured such that components of a fluidic medium in the isolation region are exchanged with components of a fluidic medium in the flow region substantially only by diffusion. In accordance with various embodiments for assessing a level of secretion of an analyte by a biological micro-object, or a population of biological micro-objects generated therefrom, using FRAP techniques, the biological micro-object is a biological cell, and wherein the method further comprises expanding the biological cell within the chamber into a clonal population of biological cells. In accordance with various embodiments for assessing a level of secretion of an analyte by a biological micro-object, or a population of biological micro-objects generated therefrom, using FRAP techniques, methods can further comprise perfusing the flow region with a culturing medium, wherein the perfusing can occur after introducing the biological micro-object into the chamber and before introducing the second fluidic medium into the flow region. In accordance with various embodiments for assessing a level of secretion of an analyte by a biological micro-object, or a population of biological micro-objects generated therefrom, using FRAP techniques, the first period of time is about 30 minutes to about 60 minutes. In accordance with various embodiments for assessing a level of secretion of an analyte by a biological micro-object, or a population of biological micro-objects generated therefrom, using FRAP techniques, methods can further include introducing a third fluidic medium into the flow region, wherein the third fluidic medium does not comprise any of the reporter molecules, and allowing at least a portion of unbound reporter molecules to diffuse out of the chamber, wherein detecting the reporter molecules located within the area of interest occurs at a time selected such that an amount of unbound reporter molecules that have diffused out of the chamber is at least 2X greater than an amount of RMSA complexes that have diffused out of the chamber. In accordance with various embodiments for assessing a level of secretion of an analyte by a biological micro-object, or a population of biological micro-objects generated therefrom, using FRAP techniques, wherein the area of interest can include at least a portion of the chamber aligned along an axis of diffusion from within the chamber to out into the flow region. The step of detecting the reporter molecules located within the area of interest can comprise measuring an intensity of a detectable signal coming from the area of interest, wherein at least some of the detectable signal emanates from the detectable label of reporter molecules located within the area of interest. In accordance with various embodiments for assessing a level of secretion of an analyte by a biological micro-object, or a population of biological micro-objects generated therefrom, using FRAP techniques, the steps of detecting the reporter molecules located within the area of interest can further comprise determining a background-subtracted signal intensity by subtracting an intensity of a background signal from the measured intensity of the detectable signal. Methods can further comprise measuring an intensity of a background signal within the area of interest, at a time prior to introducing the biological micro-object into the chamber. The measured intensity of the detectable signal or the background-subtracted signal intensity can be normalized for a number of cells observed within the chamber.

In accordance with various embodiments for assessing a level of secretion of an analyte by a biological micro-object, or a population of biological micro-objects generated therefrom, using FRAP techniques, methods can further comprise quantifying the level of secretion of the analyte. Methods can further comprise providing a secretion score for the chamber. The secretion score can be determined according to the method of any one of embodiments 12 to 25 (i.e., from the Recitation of Selected Embodiments below). In accordance with various embodiments for assessing a level of secretion of an analyte by a biological micro-object, or a population of biological micro-objects generated therefrom, using FRAP techniques, the binding component of the reporter molecule can comprise a peptide or protein. The binding component of the reporter molecule can comprises a peptide having the sequence of any one of SEQ ID NOs: 1 to 10. The binding component of the reporter molecule can comprises protein A, protein G, or an IgG-binding fragment of protein A or protein G. In accordance with various embodiments for assessing a level of secretion of an analyte by a biological micro-object, or a population of biological micro-objects generated therefrom, using FRAP techniques, the analyte secreted by the biological micro-object can be an antibody. The analyte secreted by the biological micro-object can be a protein other than an antibody. In accordance with various embodiments for assessing a level of secretion of an analyte by a biological micro-object, or a population of biological micro-objects generated therefrom, using FRAP techniques, the microfluidic device can comprise a plurality of chambers, wherein a biological micro-object is introduced into at least two chambers of the plurality, and wherein the remainder of the method is carried out with respect to each of the at least two chambers. Methods can further comprise comparing a level of secretion for chambers of the at least two chambers of the plurality. In accordance with various embodiments for assessing a level of secretion of an analyte by a biological micro-object, or a population of biological micro-objects generated therefrom, using FRAP techniques, the area of interest can comprise an image area corresponding to an area within the chamber that is most sensitive for measuring analyte concentration fluctuations, is least sensitive to the position of biological micro-objects in the chamber when analyte fluctuations are measured, and extends along an axis of diffusion between the chamber and the flow region. Referring now to Figure 32, in accordance with various embodiments, a method for FRAP is provided. In Part A of Figure 32, reagent is allowed to equilibrate across a chamber. In Part B of Figure 32, localized photobleaching is induced using, for example, structure light. As illustrated in this Figure, the localized area is box 3540. In Part C of Figure 32, partial recovery has been observed as unbleached, free reagent enters the localized area 3550. As discussed above, free reagent generally recovers quickly because of its fast diffusion rate. In Part D of Figure 32, full recovery is observed as unbleached, bound complex enters the localized area (not pictured as equilibrium has been reached, thus eliminating any observable localized area). As discussed above, bound complex generally recovers slowly (slower than free reagent) due to its slower diffusion rate and generally higher molecular weight. FRAP has many potential advantages. For example, FRAP can allow for repeated assaying or at any time reagent is equilibrated. FRAP could be performed faster than typical diffusion gradient techniques. A FRAP user could acquire repeated time series secretion measurements to resolve biological spiking noise over time, thus potentially leading to improved biological noise statistics with fewer cells and shorter culture time. The concentration measurement would be independent both within and between fields of view because the technique measures the relative magnitude of the fast and slow recovery processes within the chamber. This should not be affected by the order in which chambers are imaged, or what part of the field of view the chamber is in. Further, for example, FRAP could be used to measure binding affinity, because the ratio described above will also be strongly affected by the affinity. In accordance with various embodiments, therefore, a method of assessing a level of secretion of an analyte by a biological micro-object, or a population of biological micro-objects generated therefrom is provided. The method can comprise introducing the biological micro-object into a chamber of a microfluidic device, wherein the microfluidic device comprises an enclosure having a flow region, wherein the chamber is fluidically connected to the flow region, and wherein the chamber contains a first fluidic medium. The method can further include allowing the biological micro-object, or the population of biological micro-objects generated therefrom, to secrete an analyte into the first fluidic medium within the chamber, and introducing a second fluidic medium into the flow region for a first period of time, wherein the second fluidic medium comprises a plurality of reporter molecules. The method can further include allowing a portion of the plurality of reporter molecules to diffuse into the chamber and bind to the analyte secreted therein, thereby producing a plurality of reporter molecule: secreted analyte (RMSA) complexes, and detecting reporter molecules located within an area of interest within the microfluidic device, wherein the area of interest includes at least a portion of the chamber. The reporter molecule (or each reporter molecule) can comprise a binding component configured to bind the secreted analyte, and a detectable label. In accordance with various embodiments, a non-transitory computer-readable medium is provided in which a program is stored for causing a computer to direct a system to perform a method for determining a quantity of analyte produced by a biological micro-object, or a population of biological micro-objects generated therefrom, using FRAP techniques. An example computer system for this is provided by the block diagram of FIG. 25 illustrating a computer system 3100, upon which embodiments of the present teachings may be implemented. Details of computer system 3100 will be provided below. In accordance with various embodiments for assessing a level of secretion of an analyte by a biological micro-object, or a population of biological micro-objects generated therefrom, using FRAP techniques, the detectable label can comprises a visible, luminescent, phosphorescent, or fluorescent label. The detectable label of the reporter molecules can be a fluorescent label, wherein said detecting the reporter molecules comprises detecting fluorescence emission from the fluorescent label of the reporter molecules within the area of interest. In accordance with various embodiments for assessing a level of secretion of an analyte by a biological micro-object, or a population of biological micro-objects generated therefrom, using FRAP techniques, methods can further comprise exposing, for a second period of time, a portion of the microfluidic device comprising the chamber to electromagnetic radiation comprising a wavelength which is capable of exciting the fluorescent label of the reporter molecules. Methods can further comprise detecting fluorescence emission within the area of interest is performed after the second period of time, detecting fluorescence emission within the area of interest is performed two or more times during a third period of time, and/or detecting fluorescence emission within the area of interest is performed substantially continuously during a third period of time. In accordance with various embodiments for assessing a level of secretion of an analyte by a biological micro-object, or a population of biological micro-objects generated therefrom, using FRAP techniques, methods can further include exposing, for a fourth period of time, a portion of the microfluidic device comprising at least a portion of the chamber to electromagnetic radiation but not the flow region, wherein the fourth period of time is sufficient to photobleach the fluorescent label of any reporter molecules present in the portion of the chamber, and detecting fluorescence emission within the photobleached portion of the chamber. The detecting fluorescence emission within the photobleached portion of the chamber can be performed after the fourth period of time, can be performed two or more times during a fifth period of time; and/or can be performed substantially continuously during a fifth period of time. Further, the detecting fluorescence emission within the photobleached portion of the chamber can occur about 5 seconds to about 20 seconds after said exposing for a fourth period of time. Moreover, the steps of exposing for a fourth period of time and detecting fluorescence emission in the photobleached portion of the chamber can be repeated one or more times. In accordance with various embodiments for assessing a level of secretion of an analyte by a biological micro-object, or a population of biological micro-objects generated therefrom, using FRAP techniques, the photobleached portion of the chamber is comprised by the area of interest.

Methods . A method for assessing a level of secretion of an analyte of a biological micro-objects, or a population of biological micro-objects generated therefrom is provided, the method including: introducing the biological micro-object into a chamber of a microfluidic device, where the microfluidic device includes an enclosure having a flow region, wherein the chamber is fluidically connected to the flow region, and wherein chamber contains a first fluidic medium; allowing the biological micro-object, or the population of biological micro-objects generated therefrom, to secrete the analyte into the first fluidic medium within the chamber; introducing a second fluidic medium into the flow region, where the second fluidic medium contains a plurality of reporter molecules, and where each reporter molecule includes a binding component configured to bind the secreted analyte; and a detectable label; allowing a portion of the plurality of reporter molecules to diffuse into the chamber and bind to the analyte secreted therein, thereby producing a plurality of reporter molecule: secreted analyte (RMSA) complexes; and detecting reporter molecules located within an area of interest within the microfluidic device, wherein the area of interest includes at least a portion of the chamber. In some embodiments, the flow region may also contain the first fluidic medium. In other embodiments, the flow region may contain a fluidic medium different from the first fluidic medium. In some embodiments, the reporter molecule may bind the secreted analyte, thereby forming a RMSA complex that may have a stoichiometry of 1:1, 2:1, 3:1, 4:1, 2:2, 4:2, and the like, of the reporter molecule: secreted analyte of the RMSA complex. In various embodiments of the method for assessing a level of secretion of an analyte, detecting reporter molecules may include detecting unbound reporter molecules as well as detecting reporter molecules that are part of RMSA complexes. In various embodiments, the chamber may have an isolation region and a connection region fluidically connecting the isolation region to the flow region, wherein the isolation region and the connection region are configured such that components of a fluidic medium in the isolation region are exchanged with components of a fluidic medium in the flow region substantially only by diffusion. In various embodiments of the method for assessing a level of secretion of an analyte, the method further includes expanding the biological micro-object within the chamber into a clonal population of biological micro-objects. In various embodiments, the method may further include perfusing the flow region with a culturing medium, where the perfusing occurs after introducing the biological micro-object into the chamber and before introducing the second fluidic medium into the flow region. In some embodiments, the culturing medium may be the same as the first medium.

In various embodiments, the culturing medium may include one or more of a soluble feeder cell component, a defined dissolved oxygen component, defined pH component, an exhausted growth medium component, and/or a soluble stimulatory component. In some embodiments, viability of the cells being cultured within the microfluidic device may be improved by including a portion of the supernatant culture medium of feeder cells that provide auxiliary biomolecules that stimulate or otherwise support the cells culturing within the microfluidic device. The feeder cells themselves may not be present within the microfluidic device but may be cultured in standard reaction vessels. Harvesting and delivery of portions of the culture medium conditioned by the presence of the feeder cells to the microfluidic device may be performed. In other embodiments, the amount of dissolved oxygen may be measured and altered as desired, which may a facile process within the microfluidic environment described here, as compared to such adjustment in culture wellplates, shake flasks and the like. In some other embodiments, the pH of the culture medium within the microfluidic environment may be monitored and altered, again a more facile process than in plasticware standardly used. In yet other embodiments, exhausted growth medium may be added to the microfluidic environment, which can act as a selection mechanism to analyze which clones within the microfluidic environment may still produce the secreted analyte more readily or may be used to approximate the scaleup environment of various types of reaction vessels, which may include wellplates, shaker flasks and bioreactors. In yet other embodiments, soluble stimulatory components such as antibodies (including but not limited to CD28), cytokines, growth factors, and the like, which may stimulate the cells within the microfluidic environment to produce more rapidly or to produce different analytes than prior to introduction of the stimulatory component. In other embodiments, one or more compounds and/or reagents configured to prevent the cells from adhering to each other and the chambers may be added to the culture medium. In some embodiments, one or more of these additions to the culture medium may confer a selection pressure on one or more of the cells within the chambers. In various embodiments, introducing the second fluidic medium into the flow region includes flowing the second fluidic medium through the flow region for a first period of time. In some embodiments, the first period of time may be based on modelling of a diffusion profile for unbound reporter molecules. In some embodiments, the first period of time may be about 30 to about 60 minutes. The method may further include introducing a third fluidic medium into the flow region, wherein the third fluidic medium does not include reporter molecules; and allowing at least a portion of unbound reporter molecules to diffuse out of the chamber, where detecting the reporter molecules located within the area of interest occurs at a time selected such that an amount of unbound reporter molecules that have diffused out of the chamber is at least 2X greater than an amount of RMSA complexes that have diffused out of the chamber. Detecting may include detecting unbound reporter molecules and detecting the reporter molecules that are part of the RMSA complexes. In various embodiments, introducing the third fluidic medium into the flow region may include flowing the third fluidic medium through the flow region for a second period of time. In some embodiments, the second period of time may be selected based on modelling of diffusion profiles for unbound reporter molecules and RMSA complexes. In various embodiments, the area of interest may include at least a portion of the chamber aligned along an axis of diffusion from within the chamber to out into the flow region. In various embodiments, detecting the reporter molecules located within the area of interest may include measuring an intensity of a detectable signal coming from the area of interest, wherein at least some of the detectable signal emanates from the detectable label of reporter molecules located within the area of interest. In some embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or more of the detectable signal emanates from the detectable label of reporter molecules located within the area of interest. In some embodiments, detecting the reporter molecules located within the area of interest further may include determining a background-subtracted signal intensity by subtracting an intensity of a background signal from the measured intensity of the detectable signal. The background signal may not be measured every time reporter molecules are detected. In some embodiments, the background signal may be pre-determined based on known/standard conditions (e.g., chip type, location of chamber in the chip, type of detectable label, components of first fluidic medium). The method may further include measuring an intensity of a background signal within the area of interest, at a time prior to introducing the biological micro-object into the chamber. In various embodiments, the measured intensity of the detectable signal or the background-subtracted signal intensity may be normalized for a number of cells observed within the chamber. In various embodiments, the method may further include quantifying the level of secretion of the analyte. Quantifying the level of the secretion of the produce may be based on any of a number of measurements, such as the measured intensity of the detectable signal or the background-subtracted signal intensity, either of which may be normalized for vignetting in the field of view. The method may further include providing a secretion score for the chamber. The secretion score may be determined according to any of the methods in the sections following which describe methods of processing the detected and/or normalized fluorescence signal.

In various embodiments, the secreted analyte may have a molecular weight at least twice as great as a molecular weight of the reporter molecules. In some embodiments, the secreted analyte may have a molecular weight at least four times greater than a molecular weight of the reporter molecules. In other embodiments, the secreted analyte may have a molecular weight at least ten times greater than a molecular weight of the reporter molecule. In various embodiments, the binding component of the reporter molecule may include at least one amino acid and/or at least one nucleic acid. In some embodiments, the binding component of the reporter molecule may include a peptide or protein. In some embodiments, the binding component of the reporter molecule may include a peptide having the sequence of any one of SEQ ID NOs: 1 to 10. In some other embodiments, the binding component of the reporter molecule comprises protein A, protein G, or an IgG-binding fragment of protein A or protein G. In various embodiments, the binding component of the reporter molecule may include an aptamer. In various embodiments, the detectable label may include a visible, luminescent, phosphorescent, or fluorescent label. In some embodiments, the detectable label may be a fluorescent label. In various embodiments, the analyte secreted by the biological micro-object may include a protein, a saccharide, a nucleic acid, an organic molecule other than a protein, saccharide, or nucleic acid, a vesicle, or a virus. In some embodiments, the analyte secreted by the biological micro-object may be an antibody. In other embodiments, the analyte secreted by the biological micro-object may be a protein other than an antibody. In various embodiments, the microfluidic device may include a plurality of chambers, where the step of disposing may include disposing a biological micro-object within at least a portion of the plurality of chambers. In various embodiments, the method may further include a step of comparing a level of secretion for each chamber of a sub-set of the chambers of the plurality of chambers. The method may further include a step of comparing scores of more than one chambers of the plurality of chambers. In some embodiments, the method may further include a step of quantifying the level of secretion. In various embodiments, the method may further include a step of selecting one or more of the plurality of chambers and exporting the biological micro-object or the population of biological micro-objects generated therefrom out of the selected one or more chambers. In various embodiments, this method allows for subcloning and comparative analysis of subclones, by further expanding and assaying the resultant subclone populations. This may be accomplished by moving selected clonal population to another set of chambers within the microfluidic device and expanding again for each individual cell of the selected population. In other embodiments, the method may further include a step of exporting the selected biological micro-object or the population of biological micro-objects generated therefrom out of the microfluidic device. In various embodiments, the step of export from either the chambers to the channel or from the chamber and/or channel out of the microfluidic device may be performed on each selected chamber individually (e.g., cells from a set of selected chambers may be exported in a series of export steps, one chamber at a time). In some embodiments, the cells which are disposed within a chamber can come from a previously assayed chamber, allowing for subcloning and comparative analysis of subclones. For example, an absolute or relative value of a specific antibody may be used to select and expand cells that produce a high volume of the specific antibody. Similarly, an absolute or relative value of a family of proteins (e.g. antibodies with an IgG domain) may be used to selected and expand cells that produce a high volume of the antibody. In some embodiments, all the cells from a chamber associated with a relative or absolute value representing the amount of a secreted analyte will be selected and expanded in the same chamber or other contained area of the chip. In other embodiments, one or more of the cells from the same chamber associated with a relative or absolute value representing the amount of a secreted analyte will be selected and expanded in different chambers. In some embodiments, the above discussed steps to generate the relative or absolute value may be repeatedly performed ( 1X, 2X, 3X, 4X, or more times) on the expanded cells. In another embodiment, application of this method may permit examination of the effects of specific conditions upon cells, with feedback from repeated assays. For example, conditions and materials more closely related to large scale production of a secreted analyte may be used, in order to find and characterize the most suitable clones for further examination. In another example, diverse stimulation protocols for B-cell antibody stimulation may be examined in a more reproducible manner, and may be assayed in order to more comparably assess the benefits of one protocol over another. In accordance with various embodiments, a method of assessing a level of secretion of an analyte by a biological micro-object, or a population of biological micro-objects generated therefrom is provided. The method can comprise introducing the biological micro-object into a chamber of a microfluidic device, wherein the microfluidic device can comprise an enclosure having a flow region, wherein the chamber is fluidically connected to the flow region, and wherein the chamber contains a first fluidic medium. The method can further comprise allowing the biological micro-object, or the population of biological micro-objects generated therefrom, to secrete an analyte comprising an exogenous tag into the first fluidic medium within the chamber, and introducing a second fluidic medium into the flow region, wherein the second fluidic medium comprises a plurality of reporter molecules. The method can further include allowing a portion of the plurality of reporter molecules to diffuse into the chamber and bind to the analyte secreted therein, thereby producing a plurality of reporter molecule: secreted analyte (RMSA) complexes, and detecting reporter molecules located within an area of interest within the microfluidic device, wherein the area of interest includes at least a portion of the chamber. The reporter molecule (or each reporter molecule) can include a binding component configured to bind the exogenous tag of the secreted analyte, and a detectable label. In accordance with various embodiments, a non-transitory computer-readable medium is provided in which a program is stored for causing a computer to direct a system to perform a method for utilizing an exogenous tag to determine a quantity of analyte produced by a biological micro-object, or a population of biological micro-objects generated therefrom. An example computer system for this is provided by the block diagram of FIG. 25 illustrating a computer system 3100, upon which embodiments of the present teachings may be implemented. Details of computer system 3100 will be provided below. In accordance with various embodiments for utilizing an exogenous tag to assess a level of secretion of an analyte by a biological micro-object, or a population of biological micro-objects generated therefrom, the chamber can have an isolation region and a connection region fluidically connecting the isolation region to the flow region, wherein the isolation region and the connection region are configured such that components of a fluidic medium in the isolation region are exchanged with components of a fluidic medium in the flow region substantially only by diffusion. In accordance with various embodiments for utilizing an exogenous tag to assess a level of secretion of an analyte by a biological micro-object, or a population of biological micro-objects generated therefrom, the biological micro-object is a biological cell, wherein the method further comprises expanding the biological cell within the chamber into a clonal population of biological cells. In accordance with various embodiments for utilizing an exogenous tag to assess a level of secretion of an analyte by a biological micro-object, or a population of biological micro-objects generated therefrom, methods can further include perfusing the flow region with a culturing medium, wherein the perfusing can occur after introducing the biological micro-object into the chamber and before introducing the second fluidic medium into the flow region. In accordance with various embodiments for utilizing an exogenous tag to assess a level of secretion of an analyte by a biological micro-object, or a population of biological micro-objects generated therefrom, introducing the second fluidic medium into the flow region can comprise flowing the second fluidic medium through the flow region for a first period of time. The first period of time can be about 30 minutes to about 60 minutes. In accordance with various embodiments for utilizing an exogenous tag to assess a level of secretion of an analyte by a biological micro-object, or a population of biological micro-objects generated therefrom, the detectable label can comprise a visible, luminescent, phosphorescent, or fluorescent label. The detectable label of the reporter molecules can be a fluorescent label, and wherein said detecting the reporter molecules comprises detecting fluorescence emission from the fluorescent label of the reporter molecules within the area of interest. In accordance with various embodiments for utilizing an exogenous tag to assess a level of secretion of an analyte by a biological micro-object, or a population of biological micro-objects generated therefrom, methods can further comprise exposing, for a second period of time, a portion of the microfluidic device comprising the chamber to electromagnetic radiation comprising a wavelength which is capable of exciting the fluorescent label of the reporter molecules. Moreover, detecting fluorescence emission within the area of interest can be performed after the second period of time, two or more times during a third period of time; and/or substantially continuously during a third period of time. In accordance with various embodiments for utilizing an exogenous tag to assess a level of secretion of an analyte by a biological micro-object, or a population of biological micro-objects generated therefrom, methods can further comprise exposing, for a fourth period of time, a portion of the microfluidic device comprising at least a portion of the chamber to electromagnetic radiation but not the flow region, wherein the fourth period of time is sufficient to photobleach the fluorescent label of any reporter molecules present in the portion of the chamber, and detecting fluorescence emission within the photobleached portion of the chamber. Moreover, detecting fluorescence emission within the photobleached portion of the chamber can be performed after the fourth period of time, two or more times during a fifth period of time; and/or substantially continuously during a fifth period of time. Even further, said steps of exposing for a fourth period of time and detecting fluorescence emission within the photobleached portion of the chamber can be repeated one or more times. The process of fluorescence recovery after photobleaching (FRAP) an area (region, area of interest, or region of interest) on a chamber for making on-chip concentration measurements (i.e., secretion rate) of secreted molecules is discussed in greater detail below. In accordance with various embodiments for utilizing an exogenous tag to assess a level of secretion of an analyte by a biological micro-object, or a population of biological micro-objects generated therefrom, methods can further comprise introducing a third fluidic medium into the flow region, wherein the third fluidic medium does not comprise any of the reporter molecules; and allowing at least a portion of unbound reporter molecules to diffuse out of the chamber, wherein detecting the reporter molecules located within the area of interest occurs at a time selected such that an amount of unbound reporter molecules that have diffused out of the chamber is at least 2X greater than an amount of RMSA complexes that have diffused out of the chamber.

In accordance with various embodiments for utilizing an exogenous tag to assess a level of secretion of an analyte by a biological micro-object, or a population of biological micro-objects generated therefrom, methods can further comprise quantifying the level of secretion of the analyte. Methods can further comprise providing a secretion score for the chamber. In accordance with various embodiments for utilizing an exogenous tag to assess a level of secretion of an analyte by a biological micro-object, or a population of biological micro-objects generated therefrom, the exogenous tag of the secreted analyte can comprise a peptide sequence. The peptide sequence can comprises a FLAG epitope, a polyhistidine sequence, a hemagglutinin (HA) epitope, or a Myc epitope. The peptide sequence can comprise the amino acid sequence (from N-terminal to C-terminal) DYKDDDDK (SEQ ID NO: 11). The peptide sequence can comprise the amino acid sequence (from N-terminal to C-terminal) HHHHHH (SEQ ID NO: 12). In accordance with various embodiments for utilizing an exogenous tag to assess a level of secretion of an analyte by a biological micro-object, or a population of biological micro-objects generated therefrom, the binding component of the reporter molecule is an antibody. The binding component of the reporter molecule can comprise a chelating agent. In accordance with various embodiments for utilizing an exogenous tag to assess a level of secretion of an analyte by a biological micro-object, or a population of biological micro-objects generated therefrom, the analyte secreted by the biological micro-object can comprise a protein. The analyte secreted by the biological micro-object can be an antibody. In accordance with various embodiments for utilizing an exogenous tag to assess a level of secretion of an analyte by a biological micro-object, or a population of biological micro-objects generated therefrom, the microfluidic device can comprise a plurality of chambers, wherein a biological micro-object is introduced into at least two chambers of the plurality, and wherein the remainder of the method is carried out with respect to each of the at least two chambers. Methods can further comprise comparing a level of secretion for chambers of the at least two chambers of the plurality. Methods can further comprise selecting one or more of the at least two chambers, and exporting one or more biological micro-objects from each of the selected chambers out of the microfluidic device. Turning now to Figure 17, a workflow 1700 is presented in Figure 17 to illustrate functions performed to quantify an amount of secreted analyte present in chambers according to some embodiments of the present disclosure. In box 1702, biological micro-objects that produce the secreted analyte are maintained in one or more chambers in the microfluidic device. For example, the biological micro-objects may be cultured within the chambers or loaded into the chambers using a variety of means including gravity and/or dielectrophoretic forces, which may be optically actuated. Each chamber may contain a single biological micro-object or a plurality of biological micro-objects. The plurality of biological micro-objects may be a clonal population of biological micro-objects (e.g. a clonal population of cells) generated therefrom the single biological micro-object or may be a heterogeneous population of biological micro-objects. In box 1704, a reporter molecule having a signal component and a binding component that binds the secreted analyte is provided to the channel and the chambers. For example, a reporter molecule may be flowed into the channel and allowed to diffuse into chambers opening to the channel. Other means of providing the reporter molecule to the channel can be used. In box 1706, the reporter molecule is allowed to diffuse within the microfluidic device (e.g. within the channel and chambers) until it reaches a steady-state concentration equilibrium in its unbound state. Depending on the molecular weight of the reporter molecule, the amount of time needed to achieve a steady-state concentration equilibrium can vary. In box 1708, the reporter molecule binds secreted analytes present in the chamber. In some embodiments, flow is resumed within the channel and unbound reporter molecule diffuses out of the chamber. In box 1710, an image is generated of the chamber(s) and channel(s) comprising the unbound reporter molecules and RMSA complexes. Depending on the signal component of the reporter molecule, it may be necessary to subject the microfluidic device to a specific light (e.g. subjecting a fluorophore to a specific frequency of light) or introduce an additional reagent to visualize the signal component. In box 1712, the image of the chamber(s) and channel(s) is analyzed to calculate an amount of the secreted analyte present in the chamber(s). In accordance with the various embodiments discussed herein, the microfluidic device can comprise a plurality of chambers, wherein a biological micro-object is introduced into at least two chambers of the plurality, and wherein the remainder of the method is carried out with respect to each of the at least two chambers. Further, an example method for clonal line development is provided, the method further comprising comparing a level of secretion for chambers of the at least two chambers of the plurality. Further, methods can include selecting a set of chambers from the plurality of chambers, wherein each chamber of the set has a score indicating that the biological micro-object, or clonal population, contained therein is a top analyte producer, exporting from the microfluidic device one or more biological micro-objects contained within each chamber of the set of selected chambers, expanding the exported one or more biological micro-objects from each chamber of the set of selected chambers in corresponding reaction vessels, and determining a level of analyte secreted in each corresponding reaction vessel, thereby determining a level of secretion for each biological micro-object or clonal population. In accordance with various embodiments, a non-transitory computer-readable medium is provided in which a program is stored for causing a computer to direct a system to perform at least part of methods for clonal line development. The system can perform at least the steps up until and including exporting from the microfluidic device the one or more biological micro-objects contained within each chamber of the set of selected chambers. An example computer system for this is provided by the block diagram of FIG. 25 illustrating a computer system 3100, upon which embodiments of the present teachings may be implemented. Details of computer system 3100 will be provided below. Now turning to Figure 18, a workflow 1800 is presented in Figure 18 to illustrate functions performed to calculate an absolute or relative value representing the amount of secreted analyte according to some embodiments of the disclosure. In box 1802, an image of the microfluidic device comprising channel(s) and chamber(s) is normalized to correct for systemic error. As discussed above, a number of different normalization algorithms may be used to correct for systemic error. In some embodiments, a gain-correction factor is used to normalize the image. In some embodiments, the amount of fluorescence signal present in the channel adjacent to a chamber is used to normalize the image. In some embodiments, an auto-fluorescence image is subtracted from the image of the microfluidic device during normalization. In box 1804, a line representing the axis of anticipated diffusion trajectory from the source of the secreted analyte within a chamber (e.g. cells within a chamber) to the channel proximal to the chamber is identified. An AOI is identified that is aligned along the axis of anticipated diffusion trajectory, and extends from within the chamber to into the channel. At least a portion of the AOI includes a region having the greatest sensitivity to signal intensity while also being insensitive to cell location within the chamber. As discussed above, the AOI and the respective region having greatest sensitivity to signal/insensitive to cell location may be determined by computationally modeling a number of different parameters including but not limited to: the geometry of the chamber, the position of the source of the secreted analyte within the chamber, the molecular weight of the secreted analyte and the presence (or absence) of a flow within the channel. In box 1806, one or more concentration values are generated based on the at AOI containing the at least a portion of the AOI that is insensitive to cell location and is most sensitive to signal variance. According to the embodiment, the concentration values may be calculated based on sampling pixels within the AOI or segmenting the AOI into groups of pixels.

In box 1808, the one or more concentration values are used to calculate a relative or an absolute value representing the amount of secreted analyte present in each chamber. As discussed above, the one or more concentration values calculated for a given chamber may be normalized based on the number of biological micro-objects (e.g. cells) present in each chamber. In some embodiments, the one or more concentration values may be used to generate a curve or other composite value representing the diffusion profile from the source of the secreted analyte to the channel. In these embodiments, a slope of a line fitted to the curve of concentration values (or other composite value) may assess a secretion score associated with chambers and may be used to assess the amount of secreted analyte present in each chamber relative to the other chambers (i.e. a relative value of the secreted analyte). In another aspect, a method of clonal line development is provided, the method including: introducing an individual biological micro-object into each of a plurality of chambers of a microfluidic device, where the microfluidic device further includes an enclosure having a flow region, and where each of the chambers of the plurality is fluidically connected to the flow region and contains a first fluidic medium; allowing each biological micro-object, or a clonal population of biological micro-objects generated therefrom, to secrete an analyte into the first fluidic medium contained in the corresponding chamber; introducing a second fluidic medium into the flow region, where the second fluidic medium includes a plurality of reporter molecules, where each reporter molecule includes a binding component configured to bind the secreted analyte; and a detectable label; allowing a portion of the plurality of reporter molecules to diffuse into each chamber of the plurality and bind to at least a portion of the analyte secreted therein, thereby producing a plurality of reporter molecule:secreted analyte (RMSA) complexes in each of the plurality of chambers; detecting, for each chamber of the plurality, an intensity of a signal emanating from a corresponding area of interest, where the area of interest includes at least a portion of the corresponding chamber, and where at least a portion of the signal emanating from the area of interest emanates from the detectable label of reporter molecules located within the area of interest; determining, for each chamber of the plurality, a score based upon the detected signal intensity emanating from the corresponding area of interest; selecting a set of chambers from the plurality of chambers, where each chamber of the set has a score indicating that the biological micro-object, or clonal population, contained therein is a top analyte producer; exporting from the microfluidic device one or more biological micro-objects contained within each chamber of the set of selected chambers; expanding the exported one or more biological micro-objects from each chamber of the set of selected chambers in corresponding reaction vessels; and determining a level of analyte secreted in each corresponding reaction vessel, thereby determining a level of secretion for each biological micro-object or clonal population. A top analyte producer may be one of the top 50% of producers. In some embodiments, a top analyte producer produces analytes at a rate amongst the top 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% producing clones or higher. Alternatively, a top producer could produce analyte at greater than a threshold amount. In various embodiments, the score can be the intensity of signal emanating from the corresponding area of interest, or it can be calculated based upon the intensity of signal emanating from the corresponding area of interest. Each chamber of the plurality may have an isolation region and a connection region fluidically connecting the isolation region to the flow region, and the isolation region and the connection region may be configured such that components of a fluidic medium in the isolation region are exchanged with components of a fluidic medium in the flow region substantially only by diffusion. In various embodiments, the method further includes expanding the individual biological micro-object within some or all chambers of the plurality into a clonal population of biological micro-objects. In various embodiments, the method further includes perfusing the flow region with a culturing medium, where the perfusing occurs after introducing the individual biological micro-objects into the plurality of chambers and before introducing the second fluidic medium into the flow region. The culturing medium may be the same as the first medium. Perfusing may be performed continuously or intermittently. In some embodiments, the culturing medium may include one or more of a soluble feeder cell component, a defined dissolved oxygen component, defined pH component, an exhausted growth medium component, and/or a soluble stimulatory component. In some embodiments, viability of the cells being cultured within the microfluidic device may be improved by including a portion of the supernatant culture medium of feeder cells that provide auxiliary biomolecules that stimulate or otherwise support the cells culturing within the microfluidic device. The feeder cells themselves may not be present within the microfluidic device but may be cultured in standard reaction vessels. Harvesting and delivery of portions of the culture medium conditioned by the presence of the feeder cells to the microfluidic device may be performed. In other embodiments, the amount of dissolved oxygen may be measured and altered as desired, which may a facile process within the microfluidic environment described here, as compared to such adjustment in culture wellplates, shake flasks and the like. In some other embodiments, the pH of the culture medium within the microfluidic environment may be monitored and altered, again a more facile process than in plasticware standardly used.

In yet other embodiments, exhausted growth medium may be added to the microfluidic environment, which can act as a selection mechanism to analyze which clones within the microfluidic environment may still produce the secreted analyte more readily or may be used to approximate the scaleup environment of various types of reaction vessels, which may include wellplates, shaker flasks and bioreactors. In yet other embodiments, soluble stimulatory components such as antibodies (including but not limited to CD28), cytokines, growth factors, and the like, which may stimulate the cells within the microfluidic environment to produce more rapidly or to produce different analytes than prior to introduction of the stimulatory component. In other embodiments, one or more compounds and/or reagents configured to prevent the cells from adhering to each other and the chambers may be added to the culture medium. In various embodiments of the method, introducing the second fluidic medium into the flow region may include flowing the second fluidic medium through the flow region for a first period of time. The first period of time may be selected based on modelling of a diffusion profile for unbound reporter molecules. In some embodiments, the first period of time may be about 30 to about minutes. In various embodiments, the method further includes: introducing a third fluidic medium into the flow region, where the third fluidic medium does not comprise reporter molecules; and allowing at least a portion of unbound reporter molecules to diffuse out of the chamber, where detecting the intensity of the signal emanating from the corresponding area of interest of each chamber of the plurality occurs at a time selected such that an amount of unbound reporter molecules that have diffused out of the chamber is at least 2X greater than an amount of RMSA complexes that have diffused out of the chamber. In some embodiments, introducing the third fluidic medium into the flow region may include flowing the third fluidic medium through the flow region for a second period of time. In some embodiments, the second period of time may be selected based on modelling of diffusion profiles for unbound reporter molecules and RMSA complexes. In some embodiments, the second period of time may be about 20 to about 50 minutes. In various embodiments of the method, the area of interest may include at least a portion of the chamber aligned along an axis of diffusion from within the chamber to out into the flow region. In various embodiments of the method, detecting the intensity of the signal emanating from the corresponding area of interest of each chamber of the plurality may include subtracting an intensity of a background signal from the measured intensity of the detectable signal to determine a background-subtracted signal intensity. The background signal may not be measured every time reporter molecules are detected. In some embodiments, the background signal may be pre- determined based on known/standard conditions (e.g., chip type, location of chamber in the chip, type of detectable label, components of first fluidic medium). In various embodiments, the method may further include measuring an intensity of a background signal within the corresponding area of interest of each chamber of the plurality, at a time prior to introducing the biological micro-objects into the chambers. In some embodiments, the measured intensity of the detectable signal or the background-subtracted signal intensity may be normalized for a number of cells observed within the corresponding chamber. In various embodiments, the scores of the plurality of chambers are determined according to any of the methods in the sections following which describe methods of processing the detected and/or normalized fluorescence signal. In various embodiments, the secreted analyte may have a molecular weight at least twice as great as a molecular weight of the reporter molecules. In some embodiments, the secreted analyte may have a molecular weight at least four times greater than a molecular weight of the reporter molecules. In other embodiments, the secreted analyte may have a molecular weight at least ten times greater than a molecular weight of the reporter molecule. In various embodiments, the binding component of the reporter molecule may include at least one amino acid and/or at least one nucleic acid. In some embodiments, the binding component of the reporter molecule may include a peptide or protein. In some embodiments, the binding component of the reporter molecule may include a peptide having the sequence of any one of SEQ ID NOs: 1 to 10. In some other embodiments, the binding component of the reporter molecule comprises protein A, protein G, or an IgG-binding fragment of protein A or protein G. In various embodiments, the binding component of the reporter molecule may include an aptamer. In various embodiments, the detectable label may include a visible, luminescent, phosphorescent, or fluorescent label. In some embodiments, the detectable label may be a fluorescent label. In various embodiments, the analyte secreted by the biological micro-object may include a protein, a saccharide, a nucleic acid, an organic molecule other than a protein, saccharide, or nucleic acid, a vesicle, or a virus. In some embodiments, the analyte secreted by the biological micro-object may be an antibody. In other embodiments, the analyte secreted by the biological micro-object may be a protein other than an antibody. In various embodiments, the reaction vessels may be wells in a well-plate, shaker flasks, or bio-reactors. The reaction vessels may have a volume greater than about 20 microliters, about 1microliters, about 1 milliliter, about 10 milliliters, about 100 mL, about 1 L, or more. A bio-reactor may have one or more of the following features: closed loop control of pH and dissolved oxygen (DO) with independent control of O2 and CO2, automated liquid handling for reactor set-up, feeds, base addition and sampling, which may more closely approximate the environment of a reactor used for mass production of a secreted analyte, which may have a volume of 20 L, 50 L, 50 gal, 200 gal, or more. The bio-reactor may have a relatively small volume such as 10 mL or 15 mL (e.g., an ambr15TM (TAP Biosystems) bioreactor). A bio-reactor may have integrated viability analysis capabilities. A workflow 1900 is presented in Figure 19 to illustrate functions performed to assess an absolute or relative value representing the amount of secreted analyte in a clonal population of cells according to some embodiments of the disclosure. At box 1902, a single cell is selected for expansion. As discussed above, a cell may be selected based on the results of an assay or the cell may be selected based on other characteristics such as a phenotype and/or morphology. At box 1904, the singleell is expanded into a clonal population of cells. In some embodiments, aspects of the clonal population of cells may be analyzed as the cells proliferate. For example, the rate of proliferation, the morphology of the cells and cell adhesion may be analyzed to assess the overall health and/or viability of the cells. At box 1906, the absolute or relative value of a secreted analyte produced by the clonal population of cells is assessed. In some embodiments, the absolute or relative value may be assessed as described above with respect to Figures 12A-C and 15. In some embodiments, the absolute or relative value may be assessed using other methods, such as those described in U.S. Patent Application No. 14/964,025, the entirety of which is herein incorporated by reference. At box 1908, one or more of the cells from the clonal population of cells may be selected based on the absolute or relative value of the secreted analyte produced by the clonal population of cells. In some embodiments, the one or more cells may be selected based on aspects of the clonal population of cells that are observed during cell proliferation as discussed above. In some embodiments, the selected cells may be exported for analysis or further expansion (e.g. expansion as a cell line to produce a secreted analyte). As discussed above, in some embodiments, the process of expanding a single cell and analyzing a clonal population for a production of a secreted analyte may be repeated to assess the absolute or relative amount of a second secreted analyte or to assess whether the single cell stably produces the secreted analyte quantified at box 1906. Absolute value of secreted analyte concentration: titration curve.In some embodiments, a theoretical model of diffusion may be used to generate an absolute value based on the one or more concentration values and/or a known quantity of the secreted analyte of a biological micro-object in one of the chambers. Depending on the embodiment, different theoretical models of diffusion may be used to calculate an absolute value of the analyte based on the one or more concentration values. Depending on the embodiment, the theoretical model may model various phenomena or evaluate different assumptions. In some embodiments, a titration curve may be used to generate an absolute value of a secreted analyte of a biological micro-object. In these instances, various known amounts of the analyte may be introduced into the microfluidic device and used to generate absolute values representing the known amounts of the analyte. The absolute values representing the known amounts of the analyte may be used to generate a titration curve demonstrating, in part, a linear relationship between the absolute values and the various known amounts of the analyte. In some embodiments, a number of absolute values corresponding to known amounts of the analyte may be generated such that the titration curve contains a "dynamic range" showing the upper and lower bounds of accurate quantification of the analyte given various system parameters (i.e. the highest and lowest amount of the analyte that produces an absolute value having a linear relationship). Depending on the embodiment, various methods of replicating an anticipated diffusion profile may be used to allow the concentration values for the known concentrations of analyte to be generated in the same manner as the analyte that is generated at a source in the chamber (e.g. by a cell in a chamber). In some embodiments, varying known concentrations of the analyte of interest are incubated with the reporter molecule. In most embodiments, the concentration of the reporter molecule will be in excess of the amount of reporter molecule necessary to bind all copies of the analyte. In some embodiments, the concentration of reporter molecule will be roughly 5-200 times the amount necessary to bind all copies of the analyte. However, this range can vary based on the binding affinity of the reporter molecule for the analyte. For example, in embodiments, where the reporter molecule has a strong binding affinity for the analyte, the concentration of the reporter molecule may range from 2-200 times the amount necessary to bind all copies of the analyte. In a specific embodiment where a FITC-labelled CPD 4 (Table 1) is used to bind IgG, the concentration of the FITC-labelled CPD 4 may range from 5-100 times the amount necessary to bind all copies of IgG. The method is not limited to the use of CPD 4 but may use any reporter molecule suitable for the diffusion assay itself. For example, fluorescently labeled CPD 1, CPD 2, CPD 3, CPD 5, CPD (Table 1) may be used to generate a titration curve, and the fluorophore may be any suitably chosen fluorophore such as Alexa Fluor® 594 or HiLyte FluorTM 555. In some embodiments, an anticipated diffusion profile may be generated by providing the unbound reporter molecule to a chamber comprising secreted analyte. Over time the reporter molecule and reporter molecule: analyte complex attains an equilibrium state across the chambers and channels of the microfluidic device after a sufficient period of time (e.g., equilibration period) over which the unbound reporter molecule and the bound reporter molecule: analyte complex attains an equilibrium. In some instances, images are taken approaching the equilibrium state and/or at the equilibrium state. Exemplary period of time or equilibration periods are 1- 3 hours. However, equilibration periods can be greater than 10 minutes, greater than 30 minutes, greater than 1 hour, greater than 1.5 hours, greater than 2 hours, greater than 2.5 hours, greater than 3 hours, greater than 3.5 hours, greater than 4 hours, or greater than 4.5 hours. Alternatively or additionally, after the RMSA complex and unbound reporter molecule attain an equilibrium state, or after an equilibration period has passed, the channels may be provided with a flow of another medium which eliminates the reporter molecule (i.e. flushes) by replacing the liquid medium in the flow region with a liquid medium that does not comprise the reporter molecule. In such an instance. As such, the RMSA complex and unbound reporter molecule from the channels and the swept regions of the chambers are flushed out of the flow region. Furthermore, the RMSA complexes and unbound reporter molecules disposed within the chamber diffuse from the chamber to the channels. However, as discussed above, the unbound reporter molecules have a higher rate of diffusion than the RMSA complex. Therefore, the unbound reporter molecules diffuse out of the microfluidic device much faster than the RMSA complexes, which have a greater mass and thus a greater radius. Since molecules with a greater radius diffuse more slowly (See Stokes-Einstein equation below) and molecules with a smaller radius diffuse more quickly, the free reagent (which has a smaller radius) will diffuse out of the pen more quicky than the RMSA complexes comprising the secreted analyte and the bound reporter.

Claims

1.What is Claimed:1. A method of assessing a relative level of secretion of an analyte across a plurality of biological micro-objects, or across populations of biological micro-objects generated therefrom, the method comprising: introducing a plurality of biological micro-objects into a plurality of chambers of a microfluidic device, wherein the microfluidic device comprises an enclosure having a flow region, wherein each chamber of the plurality of chambers is fluidically connected to the flow region, and wherein the plurality of chambers contains a first fluidic medium; allowing the plurality of biological micro-objects, or the populations of biological micro-objects generated therefrom, to secrete a soluble analyte into the first fluidic medium within the plurality of chambers; introducing a second fluidic medium into the flow region, wherein the second fluidic medium comprises a plurality of soluble reporter molecules, and wherein each reporter molecule comprises: a binding component configured to bind the secreted analyte; and a detectable label; allowing a portion of the plurality of soluble reporter molecules to diffuse into the plurality of chambers and bind to the soluble analyte secreted therein, thereby producing a plurality of soluble reporter molecule:secreted analyte (RMSA) complexes; and detecting the soluble reporter molecules located within an area of interest within the microfluidic device.

2. The method of claim 1, wherein each chamber of the plurality of chambers comprises an isolation region isolated from secondary flow of the second fluidic medium in the flow region.

3. The method of claim 1, wherein each chamber of the plurality of chambers comprises a connection region isolated from direct flow of the second fluidic medium in the flow region.

4. The method of claim 1, wherein an individual biological micro-object of the plurality of biological micro-objects is disposed into an individual chamber of the plurality of chambers.

5. The method of claim 1, wherein the concentration of soluble reporter molecules is from about 1-10 times a K D of the soluble reporter molecules for the analyte. 2

6. The method of claim 1, wherein the second fluidic medium in the flow region is refreshed at a refresh rate from about 0.1-10 seconds, and the refresh rate is a duration of time to replace an entire volume of fluid in the flow region.

7. The method of claim 1, wherein allowing the portion of the plurality of reporter molecules to diffuse into the plurality of chambers comprises allowing the plurality of reporter molecules to attain an equilibrium state across the flow region and the plurality of chambers.

8. The method of claim 7, wherein the equilibrium state is attained within hours from the introduction of the second fluidic medium.

9. The method of claim 1, further comprising: obtaining a level of secretion or secretion score for each of the plurality of chambers; and ranking the plurality of chambers based on the obtained secretion scores.

10. The method of claim 1, wherein the plurality of reporter molecules comprises at least two different reporter molecules, each having a different label.

11. The method of claim 1, wherein each chamber comprises a single opening to the flow region.

12. The method of claim 1, wherein the flow region comprises a microfluidic channel and each chamber comprises an isolation region and a connection region fluidically connecting the isolation region to the microfluidic channel, wherein the connection region has a proximal opening to the microfluidic channel and a distal opening to the isolation region.

13. The method of claim 1, wherein the area of interest is at least partially within an isolation region of the chamber.

14. The method of claim 1, wherein the area of interest is within a hook region of the chamber.

15. The method of claim 1, wherein the area of interest is proximal to a region in which a biological micro-object of the plurality of biological micro-objects is located. 2

16. The method of claim 1, wherein the area of interest is along an axis of diffusion between the plurality of chambers and the flow region (e.g., along an axis of diffusion between the isolation region of the plurality of chambers and the microfluidic channel, such as along an axis of diffusion defined by the connection region).

17. The method of claim 1, wherein the area of interest is not along an axis of diffusion between the plurality of chambers and the flow region (e.g., not along an axis of diffusion between the isolation region of the plurality of chambers and the microfluidic channel, or not along an axis of diffusion defined by the connection region).

18. The method of claim 1, wherein the area of interest comprises an image area corresponding to an area within a chamber of the plurality of chambers that is most sensitive for measuring analyte concentration fluctuations, and least sensitive to the position of biological micro-objects in the chamber when analyte fluctuations are measured.

19. The method of claim 1, wherein detecting reporter molecules located within the area of interest within the microfluidic device is performed after the diffusion of reporter molecules into the plurality of chambers and the binding of reporter molecules to the analyte secreted therein is at or near steady state conditions.

20. The method of claim 1, further comprising: introducing a third fluidic medium into the flow region, wherein the third fluidic medium does not comprise reporter molecules; and allowing at least a portion of unbound soluble reporter molecules to diffuse out of the plurality of chambers. 2

21. The method of claim 1 or 20, wherein the plurality of soluble reporter molecules from the second fluidic medium being a first plurality of soluble reporter molecules, and prior to introducing the plurality of biological micro-objects into the plurality of chambers of the microfluidic device, the method further comprising: introducing a fourth fluidic medium into the flow region, wherein the fourth fluidic medium comprises a second plurality of soluble reporter molecules; acquiring an image of the microfluidic device; and introducing a fifth fluidic medium into the flow region, wherein the fifth fluidic medium does not comprise soluble reporter molecules.

22. The method of claim 21, wherein the image being a first image acquired at t = 0, the detecting of the soluble reporter molecules comprises acquiring a second image at t = 1, wherein at t = 1 the diffusion of unbound soluble reporter molecules between the flow region and the plurality of chambers has approached a steady state.

23. The method of claim 22, wherein the area of interest comprises at least a portion of a connection region of each chamber of the plurality of chambers, the method further comprising: subtracting at least the first image from the second image to produce a corrected image; and analyzing the corrected image to determine a relative amount of soluble RMSA complexes in the area of interest comprising at least a portion of each connection region for at least a portion of the plurality of chambers.

24. The method of claim 23, wherein the relative amount of soluble RMSA complexes in the area of interest is used to generate a linear approximation of changes in the amount of soluble RMSA complexes, and a slope of the linear approximation is used to compare production of secreted analyte between chambers.

25. The method of claim 24, further comprising: acquiring a background image prior to introducing the fourth fluidic medium; and obtaining the corrected image by subtracting at least the first image and the background image from the second image. 2

26. The method of claim 22, wherein the area of interest is a hook region of each chamber of the plurality of chambers, the method further comprising: subtracting at least the first image from the second image to produce a corrected image; and analyzing the corrected image to quantify a relative amount of soluble RMSA complexes in the hook region for at least a portion of the plurality of chambers, wherein the relative amount of soluble RMSA complexes in the hook region is used to compare production of soluble secreted analyte across chambers.

27. The method of claim 26, further comprising: acquiring a background image prior to introducing the fourth fluidic medium; and obtaining the corrected image by subtracting at least the first image and the background image from the second image.

28. The method of claim 21, wherein the first plurality and the second plurality of soluble reporter molecules comprises a same label.

29. The method of claim 21, wherein the first plurality and the second plurality of soluble reporter molecules comprises two different reporter molecules each having a different label.

30. The method of claim 21, wherein the image being a first image acquired at t = 0, the method further comprising: acquiring a second image of the microfluidic device, before introducing the third fluidic medium into the flow region and after allowing the portion of the plurality of soluble reporter molecules to diffuse into the plurality of chambers and bind to the soluble analyte secreted therein.

31. The method of claim 30, wherein the detecting of the reporter molecules comprises acquiring a third image of the microfluidic device. 2

32. The method of claim 31, wherein the area of interest comprises at least a portion of a connection region of each chamber of the plurality of chambers, the method further comprising: subtracting at least the first image from the third image to produce a corrected image; and analyzing the corrected image to quantify an amount of soluble RMSA complexes in each of a plurality of sub-regions of each connection region for at least a portion of the plurality of chambers, wherein the amount of soluble RMSA complexes in a portion of the plurality of sub-regions are used to generate a linear approximation, and a slope of the linear approximation is used to compare production of secreted analyte between chambers.

33. The method of claim 32, further comprising: acquiring a background image prior to introducing the fourth fluidic medium; and obtaining the corrected image by subtracting at least the first image and the background image from the third image.

34. The method of claim 32, further comprising: acquiring a background image prior to introducing the fourth fluidic medium; and obtaining the corrected image by subtracting at least the first image, the second image, and the background image from the third image.

35. The method of claim 31, wherein the area of interest is a hook region of each chamber of the plurality of chambers, the method further comprising: subtracting at least the first image from the third image to produce a corrected image; and analyzing the corrected image to quantify an amount of RMSA complexes in the hook region for at least a portion of the plurality of chambers, wherein the amount of RMSA complexes in the hook region is used to compare production of secreted analyte between chambers.

36. The method of claim 35, further comprising: acquiring a background image prior to introducing the first fluidic medium; and obtaining the corrected image by subtracting at least the first image and the background image from the third image. 2

37. The method of claim 35, further comprising: acquiring a background image prior to introducing the first fluidic medium; and obtaining the corrected image by subtracting at least the first image, the second image, and the background image from the third image.

38. The method of claim 30, wherein the second image is used to determine whether a diffusion of unbound reporter molecules between the flow region and the plurality of chambers has reached a steady state.

39. The method of claim 1, wherein the secreted analyte has a molecular weight at least twice as great as a molecular weight of the soluble reporter molecules.

40. The method of claim 1, wherein the secreted analyte has a molecular weight at least four times greater than a molecular weight of the soluble reporter molecules.

41. The method of claim 1, wherein the secreted analyte has a molecular weight at least ten times greater than a molecular weight of the soluble reporter molecule.

42. The method of claim 1, wherein the analyte secreted by the biological micro-objects comprises a protein, a saccharide, a nucleic acid, an organic molecule other than a protein, saccharide, or nucleic acid, a vesicle, or a virus.

43. The method of claim 1, wherein the analyte secreted by the biological micro-object is an antibody or, optionally, a glycosylated antibody.

44. The method of claim 1, wherein the binding component of the reporter molecule binds to an antibody heavy chain H1 domain.

45. The method of claim 1, wherein the binding component of the reporter molecule binds to an antibody light chain (e.g., an epitope that includes one or more amino acid residues of a light chain peptide).

46. The method of claim 1, wherein the binding component of the report molecules binds to a lambda light chain (e.g., an epitope that includes one or more amino acid residues of a lambda light chain peptide). 2

47. The method of claim 1, wherein the binding component of the report molecules binds to a kappa light chain (e.g., an epitope that includes one or more amino acid residues of a kappa light chain peptide).

48. The method of claim 1, wherein the microfluidic device comprises a plurality of chambers, wherein a biological micro-object is introduced into each of at least two chambers of the plurality, and wherein the remainder of the method is carried out with respect to each of the at least two chambers.

49. The method of claim 1, further comprising: comparing a level of secretion for chambers of the at least two chambers of the plurality of chambers.

50. The method of claim 49, further comprising: comparing secretion scores of more than one chamber of the plurality of chambers.

51. The method of claim 1, wherein introducing the plurality of biological micro-objects into the plurality of chambers of the microfluidic device comprises selecting the biological micro-objects and selectively moving the selected biological micro-objects into the plurality of chambers.

52. The method of claim 51, wherein the selection is a positive selection.

53. The method of claim 51, wherein the selection is a negative selection. 2

54. A method of clonal line development, comprising: performing the method of any one of claims 1-53; selecting a set of chambers from the plurality of chambers, wherein each chamber of the set has a score indicating that the biological micro-object, or clonal population, contained therein is a top analyte producer; exporting from the microfluidic device one or more biological micro-objects contained within each chamber of the set of selected chambers; expanding the exported one or more biological micro-objects from each chamber of the set of selected chambers in corresponding reaction vessels; and determining a level of analyte secreted in each corresponding reaction vessel, thereby determining a level of secretion for each biological micro-object or clonal population. For the Applicant WOLFF, BREGMAN AND GOLLER by: